11alpha-hydroxylation of steroids by glomerella



United States Patent Ofilice 2,985,563 Patented May 23, 1961 Ila-HYDROXYLATION OF STEROIDS BY GLOMERELLA Fernando Carvajal, Ridgewood, NJ., assignor to Schering Corporation, Bloomfield, N.J., a corporation of New Jersey No Drawing. Filed Nov. 13, 1958, Ser. No. 773,579

51 Claims. (Cl. 195-51) The present invention relates to the microbiological treatment of steroids to eifect a selective chemical modification thereof. More particularly, this invention relates to the eifective and substantial microbiological transformation of eleven desoxy steroids to form the corresponding ll-hydroxy derivatives thereof.

Heretofore it has been known to introduce oxygen into the eleven position of a steroid nucleus. Among the processes suggested for accomplishing this oxidation, the biochemical have been of particular interest, since it has made possible the direct introduction of oxygen at this point, avoiding the plurality of steps inherent in the highly involved organic syntheses, otherwise necessary. Bio-oxidation is accomplished by subjecting a steroid containing an eleven methylene group to the action of oxygenating enzymes produced by various microorganisms. Such biooxidative techniques have hitherto presented certain difficulties. Thus, by way of illustration, a number of oxidation products may result which are only diificultly separated, and, in any event, necessitate the inclusion of an added step in the process. Further, the yield of product, such as ll-epi-hydrocortisone, expressed as a proportion of the starting material such as Substance S, may be significantly small. Similarly, and most significantly, the concentration of steroid substrate and necessary period of time required to effect even a reduced yield of oxygenated product by certain of the known bio-oxidative procedures would be such as to render these techniques prohibitive. Also certain of the known hydroxylating microorganisms are so diflicultly developed and sustained as to seriously limit their commercial feasibility. Hence, the process of the present invention is believed to present a significant advance over methods previously proposed, since it pre sents a procedure whereby significantly great concentrations of an II-desoxy steroid substrate undergoes a substantial and often complete conversion to its corresponding ll-hydroxylated derivatives.

Accordingly, the present invention provides a novel, eflicient and expeditious procedure for the production of ll-hydroxylated steroids, which is of material significance commercially, and which comprises subjecting eleven desoxy steroids to the oxidative action of an easily developed and sustained hydroxylating fungus of the class Ascomycetes, order Sphaeriales, family Sphaeriaceae, genus Glomerella, and particularly, and indeed most desirably, those isolates of the species cingulata, or oxidizing enzymes thereof. Glomerella cingulara, and particularly one of the following strains thereof, i.e. ATCC 10529, ATCC 10530, ATCC 10531, ATCC 10532, ATCC 10533, ATCC 10534 and QM 1407, is preferred as the oxidative fungus. It should be recognized that while Glomerella cingulata and most desirably the strain ATCC 10534 is advantageous particularly in the lle-hydroxylation of 11- desoxy l6a-alkyl-4-pregnenes (e.g. l6e-methyl 4-preg nene-l7u,21-diol-3,20-dione), ll-desoxy 16a alkyl 1,4- pregnadienes (e.g. l6m-methyl l,4-pregnadiene-l7a,21- diol-3,20-dione), and the corresponding non-alkylated 11- desoxy 4-pregnenes (e.g. 4-pregnene 17oz,21-di0l-3,20-

dione) and 1,4-pregnadienes (cg. 4-pregnene :,21- diol-3,20-dione), other members of the genus Glomerella, such as for example, G. fusarioia'es (ATCC 9552), and the following (obtainable from the Centraal Bureau voor Schirnmelcultures) G. lagenarium, G. major, G. phat-idiomorphat, G. rubz'cola are operative for the production of oxygenated steroid and possess a distinctive physiological specificity which will transform ll-desoxy steroids to the corresponding ll-epimeric steroids.

These fungal organisms including their mutants are stable and easily grown and can be obtained, as indicated, from known sources, such as the American Type Culture Collection (ATCC), Washington, D.C.; the Quarter Master Culture Collection, Natick, Massachusetts (QM); or the Centraal Bureau voor Schimmelcultures, Baarn, Holland. Alternatively, they can be obtained from natural sources using techniques known to microbiologists.

Representative of the ll-desoxy steroids for use in the practice of my invention are: 4-pregnene-l7u,2l-diol-3,20- clione (ll-desoxy-l'ia hydroxy-cortisone, Reichstein's Substance S), 1,4-pregnadiene 17a,21-di0l 3,20-dione (l-dchydro 11 desoxy-17a hydroxycortisone), 16ozmethylallopregnane 1711,21 diol 3,20-dione, 16B- methylallopregnane 17,21 diol 3,20-dione, 16a-thulvlallopregnane 1711,21 diol 3,20-d1'one, Ion-ethylalloprcgnane 17a,2l diol-3,20-dione, IGfl-methyl 4- pregnene 1711,21 diol 3,20-dione, lfia-methyl 1,4- prcgnadiene 170:,21 diol -3,20-dione, 16fi-methyl-L4- pregnadiene 17:1,21 diol 3,20-dione, l6a-t-butyl-l,4- pregnadiene 1701,21 diol 3,20-dione, lfifi-ethyl-IA- pregnadiene 1711,21 diol 3,20-dione, pregnane-l7a,2ldiol 3,20 dione, allopregnane l7a,2l-diol-3,20-dione, pregnane 1701,21 diol 3,20dione, 21-acetate, 16amethyl 4 pregnene 170:,21 diol-3,20-dione, 1,4,6- pregnatriene 17a,2l diol 3,20-dione, as well as the 21- acyl (eg. acetate, propionate) derivatives and particularly the acyl derivatives of the lower alkanoic acids thereof. It will be apparent that the ll-desoxy steroids which can be hydroxylated at the ll-carbon atom of the steroid nucleus by the process herein described are not narrowly delimited, but encompass those ll-desoxy compounds having a cyclopentanoperhydrophenanthrene nucleus e.g. 11- desoxy sterols, bile acids, cardiac aglycones, saponins, sex hormones. D-vitamins and the like and particularly those wherein the A-ring contains preferably a 3-keto configuration and a cortical side chain containing 2 carbon atoms attached to the D-ring at 0-17. In addition, it is often desirable that at least one double bond be present (e.g. at A in the A-ring). In general, however, the more preferred compounds for use in the practice of my invention are the ll-desoxy-pregnanes, allopregnanes, and unsaturated analogues thereof (including within the term pregnanes and allopregnanes as employed herein, and unless otherwise explicitly indicated, the substituted derivatives thereof as well), and particularly those compounds represented by the structure:

CHIOR l-pregnene, 4-pregnene, 1,4-pregnadiene and 1,4,6-pregnatriene analogues thereof.

The product in each instance is the corresponding 11a.- hydroxy compound, which is formed in high and in some instances quantitative yield. In particular, Reichsteins Substance S, and its 16a-methyl derivative are converted to 4-pregnene-l 1:1,17 e,2l-t:riol-3,20-dione and lfia-methyl- 4-pregnene-llu,l7a,2l-triol-3,20-dione respectively in yields of excess of 75%. It should be noted that where the steroid substrate contains a -21 acyloxy radical. this group is generally hydrolyzed to the corresponding C-Zl alcohol by Glomerella simultaneous with introduction of the llu-hydroxy group.

Examples of other less preferred compounds which can be hydroxylated in the Ila-position by the process of the present invention include ldfi methyl-S-pregnen-lifi,17adiOI'ZO-OIIC, 35,2 1 -diacetoxyl6oz-methyl-5-pregnene-3B, l7a-diol-20-one] and lSfi-methyl-S-pregnene-Bfl,I7m-diol- -one, the preparation of which is described hereinafter, as well as the corresponding l6-ethyl, l6-tert. butyl and like homologues thereof.

The microorganism described herein can also effect dihydroxylation with steroid substrates other than those described hereinabove. Thus, for example, 4-pregnene- 3,20-dione (progesterone) can be transformed by our procedure in high yields to the corresponding 11.15- dihydroxy progesterone together with minor amounts of the corresponding 6,1 l-dyhydroxy progesterone.

The ll-desoxy and ll-oxygenated steroids disclosed herein containing alkyl substituents in the C-l6 position together with processes for their production are the invention of Eugene P. Oliveto and Richard Rausser and are not claimed in the present application apart from my invention which is limited to a novel process wherein lldesoxy steroids are transformed by species of fungus of the genus Glomerella to the corresponding lla-hydroxylated derivatives.

A variety of procedures can be employed to prepare the ld-alkyl steroids. Illustratively, 16aor l6g9-alkyl-1ldesoxyallopregnanes can be prepared from la-alkyl or l6fi-alkyl pregnenolones, such as for example, 16!:- methyl pregnenolone, 16 S-tert.-butylallopregnenolone and the like by initially hydrogenating the IG-alkyl-pregnenolone over such conventional catalytic agents as palladium on charcoal to cause the formation of a solid precipitate, the lfiaor l6,6-alkylallopregnane-3B-ol-ZO- one, e.g. 1611- or 16fl-methylallopregnane-36-ol-20-one. In order to introduce an hyroxyl group at C-17, the C-20 ketogroup of this latter compound is converted to the cool-acetate by refluxing said l6alkylallopregnane-3B- ol-20-one with acetic anhydride and a strong acid, such as p-toluenesulfonic acid or perchloric acid, for example. It is preferred not to isolate the enol-acetate but rather to react said substance in situ with a peroxy-acid, such as for example, peracetic acid, perbenzoic acid, monoperphthalic acid, or pertrifluoroacetic acid, whereby a second intermediate, a 17,20-epoxide, preferably not isolated. is obtained. Treatment of the reaction mixture with alkali hydrolyzes the epoxide in situ to cause the production of the crude. solid, l7a-hydroxylated product which can be separated out suitably by filtration and subsequent crystallization from a medium such as methanol-water. The crystalline product is l6-alkylallopregnane-318,170:- diol-ZO-one. Acyloxylation, e.g. acetoxylation, of the C-2l methyl group is effected in the conventional manner as noted hereinabove, such as by bromination of the C-21 methyl group, followed by reaction of the bromoderivative with, for example, sodium or potassium acetate, whereby l6e-alkylallopregnane-3B,l7a,21-triol-20 one ZI-acetate is formed.

The product so obtained is oxidized sequentially at the 3-C position by known standard procedures such as for example with N-bromoacetarnide, N-bromosuccinimide or chromic acid toyield upon subsequent crystallization from acetone-hexane, lfi-alkyiallopregnane-l7e,2l-diol- 4 l 3,20-dione ZI-acetate. This latter compound can be saponified so as to produce the corresponding 0-21 alcohol, 16-alkylallopregnane-l7a,21-diol-3,20-dione, by reaction with hydrolytic agents such as aqueous methanolic potassium bicarbonate or with such reagents as sodium carbonate, sodium hydroxide, sodium alkoxides (e.g. sodium methoxide, sodium ethoxide) and acids such as p-toluenesulfonic acid.

Microorganisms such as for example F [avobacterium dehydrogenans var. hydrolyticum can also be employed for this purpose using standard procedures such as that disclosed in Union of South Africa Patent 3,462/55 and described in detail hereinafter. The crude product thus formed is desirably separated out by filtration and crystallized from acetQnehexane. The one hydroxyl group is then, if desired. introduced into the Ila-position of said l6-alkylal1oprcgnanc-l7m,2ldiol-3,20-dione by the proccss of my invention employing a microorganism of the genus Glornerella.

The resultant products are valuable intermediates in the formation of the corresponding llfi-hydroxy and iiket0-l,4-dienes which are valuable therapeutically active compounds useful in the treatment of inflammatory diseases such as arthritis. To effect the production of the lla-hydroxy 1,4-dienes the 16a-alkylallopregnane-11e, l7a,2l--triol-3,2D-dione is initially reacted with chromium trioxide in pyridine to cause the oxidation of the 11m hydroxy to the ll-keto derivative, l6a-alkylallopregnane- 17a,21di0i-3,11,20-ill0l16. This compound is then converted to the corresponding 1,4-diene by dihalogenation, preferably dibromination which occurs at the Z-carbon and 4-ca1'bon linkage of the A-ring. Didehydrobromination is then effected with a basic agent, preferably dimethylformamide, to produce the desired 1,4-diene, 16aalkyl-l,4-pregnadiene-l7a,2l-diol-3,l1,20-trione.

Alternatively, the l6-alkylallopregnane-l7a,2l-diol-3, ZO-dione ZI-acylate can be dehydrogenated initially in the A-ring in order to eiiect the preparation of the corresponding ll-desoxy 1,4-diene by the procedure described immediately above whereby the l6-alkylallopregnanel7e,21-diol-3,20-dione Zl-acetate is dihalogenated prefen ably with bromine, to form the intermediate 2,4- dibromol fi-alkylallopregnanel 7e,2 l -diol-3,20-dione 2 lacetate. Didehydrobromination with basic agents, preferably dirnethylformamide, produces the Zl-acetate of i6- alkyl-1,4-pregnadiene-17a,2l-diol-3,20-di0ne, which can then be saponificd so as to produce the corresponding C-Zl alcohol, l-alkyl-1,4-pregnadiene-l7a,2l-diol-3,20- dione, by employment of standard hydrolytic agents such as those described above. The hydroxyl group is then introduced into the lie-position thereof by the process of my invention as described in detail hereinafter employing a microorganism of the genus Glomerella. The resultant 16 alkyl 1,4-pregnadiene-lla,l7a,21-triol-3,20- dione can, of course, be again esterified at the C-21 position if desired by standard acyloxylation, e.g. alkanoyloxylation, procedures such as that noted hereinabove, e.g. reaction with acetyl chloride and pyridine and sequentially oxidized with chromic acid to the corresponding ll-keto derivative, 16-alkyl-1,4-pregnadiene-l7e,2ltriol-3,11,20trione which is a valuable therapeutic agent in the treatment of anti-inflammatory disease, such as arthritis.

Further, 16,3-alky1-17a-hydroxy-4-pregnenes for use in the process of my invention can be prepared in the following manner wherein a l6-a1kyl5,16-pregnadiene3B-ol- ZO-one is employed as the starting material. This compound is initially epoxidized at the l6(17)-linkage of the D-ring, with hydrogen peroxide. The 3-C hydroxyl group of this epoxy compound is then acetylated with acetic anhydride in a basic reaction medium, e.g. pyridine, to yield 3fl-acetoxy-lfifl-methyl-lfia,l7a-epoxy-5 pregnene- 20-one. Although acetylation is shown and is indeed the preferred procedure, other acylations such as for example with propionic anhydride to cause the formation of the corresponding 3fi propionoxy derivative can also be performed.

The Bti-alkanoyloxy comp: und thus produced is then reacted with glacial acetic acid and a hydrogen halide, e.g. hydrogen chloride, hydrogen bromide, to effect the preparation of the alkylene derivative, for example, the 16-methylene compound, Syd-acetoxy-l6-rnethylene-5-pregnene-l7a-ol-20-one, which is hydrogenated by a standard procedure using, for example, palladium on charcoal, to form the corresponding l6-alkyl derivative, e.g. lie-acetoxy-1G-fi-methyl-S-pregnene-l7ot-ol-20-one. This compound can then be acyloxylated in the C-21 position of the D-ring by halogenation, preferably bromination, of the C-21 methyl group, followed by reaction of the bromoderivative, thus produced with sodium or potassium acetate, butyrate or the like, in the usual manner, to give the 33,21-dialkanoyloxy-16,8-alkyl-5-pregnene-l7ot-ol-20- one. This compound when treated with a hydrolytic agent, e.g. sodium bicarbonate, sodium hydroxide, toluenesulfonic acid, followed by oxidation of the 3-hydr0xyl group, as by chromic acid in acetone, and rearrangement of the 5,6-double bond to 4,5 by the action of acid or when treated with a microorganism such as Flavobacterium dehydrogenans var. hydrolyticum will yield lofi-alkyll7a,2l-dihydroxy-4-pregnene-3,ZO-dione. Subsequent exposure to the enzymatic activity of a strain of Glomerella in accordance with the practise of my invention will result in the introduction into said l6fi-alkyl-l7a,2l-dihydroxy-4-pregnenc-3,20-dione of the lla-hydroxyl group. These resultant compounds, the 16,8-alkyl-l1a,17a,2l-trihydroxy-4-pregnene-3,20-dioncs are valuable intermediates in the formation of the corresponding ll5-hydroxy-4- monoenes and the ll-keto 4-monoenes which compounds are therapeutically useful in the treatment of inflammatory diseases, such as for example, arthritis, or can be converted to the corresponding 1,4-dienes, 16,6-alkyl-lla,17a,2l-trihydroxy-1,4-pregnadiene-3,20-diones by reaction with Corynebacterium simplex according to procedures de scribed in United States Patent 2,837,464; these compounds can then be readily converted to the corresponding llp-hydroxyl or ll-keto-1,4-dienes which are therapeutically active anti-inflammatory compounds useful in the treatment of arthritis and like diseases.

Similarly, l6ot-alkyl-l7ahydroxy-4-pregnenes can be prepared from l6a-alkyl-pregneno1one-3-acetate (eg. 160:- methyl-5-pregnene-3fi-ol-ZO-one 3-acetate as well as the corresponding propionate, butyrate or the like), by sequentially halogenating e.g. chlorinating this latter compound in the 5-C and 6-C positions, in a basic medium such as pyridine, lutidine or the like; introducing the by droxy group into the l7-C position by the method described above, that is by enolacetylation of the 20-C keto group with acetic anhydride and p-toluenesulfonic acid or the like and sequential reaction of this ZO-enOl-acetate, preferably in situ, with an acid such as peracetic acid or perchloric acid to form the corresponding 17,20-epoxide. This compound too is treated in situ with alkali, e.g. potassium bicarbonate, and hydrolyzed thereby to yield the 16u-alkyl-5,6-dichloropregnane-3,l7a-diol-20-one 3 acetate. Acyloxylation of the C-21 position is then effected in the standard manner by bromination of the 0-21 methyl group and subsequent reaction thereof with so dium or potassium acetate or the like. The resulting compound, 16a-alkyl-5,6-dichloro-pregnene-3fi,17a,2l-triol-20 one ZI-acetatc, is then oxidized at the C-3 position of the A-ring and hydroyzed at C-2l by means of a microorganism, e.g. F lavobacterium dehydrogenans var. hydrolyticum, as noted above, to yield l6ct-alkyl-5,6-dichloro-pregnane- 17a,21-dl0l3,20di0ll. This latter compound is readily dehalogenated employing well-known procedures e.g. zinc dust in an acid medium such as acetic acid, which results in the formation of a double bond at the 4(5)-carbon linkage, the product being l6m-alkyl-4-pregnene-l7a,2ldiol-3,20-dione 2l-acetate.

Alternatively, l6u-alkyl-4-pregene-l7a,2l-diol-3,20-dione is prepared from l6a-alkyl-5,6-dichloropregnane- 3,17a,21-triol Zl-acetate, preferably, by reaction thereof with nascent chromous chloride to form l6u-alkyl-5- pregnene-3B,17,2l-triol-20-one ZI-acetate which is transformed to the corresponding 4-pregnene with Flavobacterium dehydrogenans var. hydro lyticum or alternatively with standard chromium trioxide reagent to form the l6a-alkyl-5-pregnene-l7u,2l-diol-3,20-dione which undergoes intramolecular rearrangement when treated with a methanolic solution of sodium bicarbonate Or the like, to produce the desired l6e-alkyl-4-pregnene-17a,2l-diol- 3,20-dione.

This compound however prepared, when subjected to the enzymatic activity of a species of the microorganism of the genus Glomerella according to the process of my invention as described herein, results in the Ila-hydroxylation thereof to produce the valuable intermediate l6a-alkyl-4-pregnene-l1a,17a,2l-triol-3,2O dione which can be converted by known procedures to the corresponding 1l-keto-4-monoenes (e.g. by oxidation with chromic acid), their corresponding 1l-keto-1,4-dienes, or if desired, the llt3-hydr0xy-4-monoenes or llB-hydroxy-IA- dienes, all of which are valuable agents in the treatment of inflammatory diseases such as arthritis, as noted above.

The hydroxylation process of my invention is conveniently accomplished by cultivating the microorganism, for example the preferred Glomerella cinglilata. unde aerobic conditions, on a suitable medium such as described hereinafter, in intimate admixture with the ll-desoxy steroid. such as Substance S, the cultivation or growth of the fungus being continued until the desired enzymatic hydroxylation is effected. Alternatively, and indeed preferably, the process of the present invention is effected by growing a microorganism in a suitable fermentation medium under aerobic conditions, and then separating the cells of the microorganism so cultivated therefrom. The lldesoxy steroid, such as for example the Substance S referred to hereinabove, which is to be hydroxylated, is then added to these cells under aerobic conditions, for a period of time sufficient to etfect the desired oxygenation. This latter procedure results in a marked simplification of the recovery step, but while the latter method is. deemed the most desirable the order of addition is not critical. Thus the ll-desoxy steroid can for example also be added to the growth medium which is then inoculated with the fungus.

The eleven desoxy steroid starting material is introduced into the nutrient medium by known standard procedures, as for example, by forming a suspension thereof in water, or by preparing a solution or suspension thereof, in a solvent such as methanol, ethanol, acetone, propylene glycol, dimethylformamide, or dimethylacetamide, or other water-miscible organic solvent which is nontoxic to the microorganism. As noted elsewhere herein exceptionally high concentrations of steroid can be effectively transformed, e.g. up to 30 g. per liter of total fermentation mix. It may, in addition. be added in a finely divided form such as a solid micronized powder. It is ordinarily preferred that the steroid be present in very finely divided form in order to expose the greatest surface area thereof and thus permit maximum contact with the oxygenating enzyme produced by the microorganism and the most efiicient conversion of eleven desoxy steroid to its eleven alpha hydroxylated derivative. Optionally, the steroid can be added at one time or introduced in a continuous or intermittent manner over a period of time.

Suitable nutrient media for the cultivation of Glomerella include assimilable carbon, organic and inorganic sources of nitrogen together with minor amounts of inorganic salts and trace elements. The concentration of these constituents can be varied within wide limits. Standard sources of carbon appropriate for use in these growth media are carbohydrates such as dextrose, glucose, starch, inverted molasses and the like. Organic nitrogen sources normally employed are such substantially proteinaceous materials as corn steep liquor, laotalbumin digest, yeast extract, or soybean meal containing from approximately ten percent to fifty percent protein. Sources of inorganic bound nitrogen are represented by ammonium nitrate, diabasic ammonium phosphate and the like. Trace elements are supplied by the inclusion in the culture medium of tap water. Inorganic salts (e.g. suitable water-soluble salts of magnesium, zinc, potassium, sodium, phosphorous, iron, and the like) and other such materials, for example, niootinamide, are normally present in the sources of assimilable carbon and organic nitrogen in amounts sufficient to assure optimum growth of the microorganism in the culture broth but can be separately added to the growth media if so desired.

The following are examples of suitable aqueous nutrient media for use in the present invention:

Medium Number l:

Yeast extract (Difco) percent l Dextrose (Cerelose) do 1 Tap water do 6.5 pH 98 Percentages as referred to throughout this specification and unless otherwise explicitly indicated, are percentages by weight.

Medium Number 2: Gm. Asparagin-e Glucose 25 KH PO 0.5 MgS0 f/H 0 0.25 Tap water to one liter.

Medium Number 3: Gm. Sodium nitrate 2 K HPO 1 MgSO .7l-l 0 0.5 Yeast extract 1 KCl 0.5 Glucose 50.0

Tap water to one liter.

The pH of the culture medium is not critical, although it is known that fungi fare better in a slightly acid medium as opposed to an alkaline medium. Thus, a pH of 4 to 8 is thoroughly operative for proper growth of Glomerella, with a preferred pH being in the range of 5.3 to 7.5. The pH is adjusted to these ranges by addition of a suitable acid or alkaline material, such as, for example, sodium hydroxide or potassium hydroxide or hydrochloric acid. The addition of small quantities of anti-foaming agents, although not essential, is desirable, particularly in commercial scale operation.

To promote the growth of Glomerella and the biochemical transformation of the steroid substrate, Substance S, the culture is preferably shaken and/or aerated and stirred.

Thus the eleven desoxy steroids employed in the present invention can be suitably oxygenated in the following manner:

The spores as received from the culture collection, or vegetative growth of an oxygenating strain of 610merella cz'ngulata suspended in 100 ml. sterile distilled water, are grown on an agar medium (e.g. 0.3 percent yeast extract, 1.0 percent Cerelose, 0.1% corn steep liquor) at a preferred temperature of 22 C. to 28 C., although temperatures as low as C. or as high. as 35 C. are not detrimental. After a period of time, about three to ten days, and preferably about seven days, sporulation occurs. The growth medium and spores are then diluted and washed with sterile water and after several washings, there remains a heavy spore suspension which is used for inoculation of the liquid fermentation medium as described above. The conversion from the spore stage to the vegetative mycelium stage generally occurs in 12 hours to 48 hours and can actually occur in as little as 3 hours. At the end of this growth period, which is measured by the appearance of a thick vegetative mycelium, an inoculum consisting of about one percent vegetative mycelium is added to shake flasks containing the fermentation medium, and the flasks are shaken on a rotary shaker until substantial growth is observed (generally 12 hours to 24 hours). At this point, the eleven desoxy steroid substrate, finely divided or dissolved as described hereinabove is added to the culture medium. The steroid concentration in an alcohol medium, for example, is normally from 2 to 3 mg. of steroid per gram of solvent. Increased concentrations of steroid in solvent are had by warming the alcohol or substituting another solvent therefor such as dimethylformamide wherein concentrations of up to mg. and more of steroid are completely dissolved. The concentration of steroid in the total fermentation mixture can be as low as 100 mg. per liter or as high as 30 g. per liter, although a concentration of 10 g. to l5 g. per liter is preferred. The fermentation mixture. is shaken until complete conversion is effected (e.g. about 6 hours to 96 hours). The conversion of eleven desoxy steroid, such as Substance S to the eleven alpha hydroxylated derivative, 4pregnenella,17n,21-triol-3,20-dione, is determined in the conventional manner by paper chromatography.

Alternatively, instead of employing a one percent inoculum of the vegetative mycelium, a spore suspension may be used directly. However, such a procedure is less preferred since it requires extended periods of time for proper growth of mycelium and conversion of eleven desoxy steroid.

At the conclusion of the fermentation process, the desired ll-hydroxylated steroid, is recovered from the fermentation medium by the following procedure, which describes in particular a 109 ml. fermentation. This is a general procedure and is operative for fermentation of various amounts.

The mixture is extracted (6. g. usually three or more times), each extraction employing, by way of illustration, two volumes of organic solvent per volume of fermentation broth. The solvent, which may be a halogenated organic solvent, e.g. chloroform, methylene chloride, an ester, e.g. ethyl acetate, an alcohol, e.g. butanol, an ether, e.g. diethyl ether, dibutyl ether, an aromatic solvent, toluene or the like, is then cooled and dried over sodium sulfate, or like standard drying agent, such as calcium chloride or magnesium sulfate, and then filtered. The filtrate is evaporated to dryness or to a small volume. This solution is used for characterization of steroid content as described hereinafter.

The foregoing growth and fermentation procedure is generally applicable to a small scale process wherein shake flasks on rotary shakers are employed and may be varied on a larger scale so that the fermentation is carried out in tanks and occurs at a much faster rate. The vegetative mycelium can be added to fresh nutrient at a concentration of from about one percent to ten percent and higher and growth is permitted to occur preferably at about 28 C. for approximately 24 hours. Lower concentration of mycelium are preferred since better aeration, a factor directly associated with more eflicient transformation is effected. In this latter instance a submerged inoculum may be employed, into which air is introduced as the oxygen supply. It is generally known that growth mixtures of this type cause the production of large quantitles of foam due to the rapid import of air and defoaming agents are therefore usually employed such as, for example, one percent octadecanol or hexadecanol in lard oil, a silicone such as GE. 60 anti-foam (a product of the General Electric Company, Schenectady, New York), a substituted oxazoline which is a non-volatile, amine-type, cationic surface active agent available under the trade name Alkaterge C or the like. In this latter procedure, when the growth period is substantially complete, the steroid substrate, e.g., Substance S, in alcohol, may be added so that its concentration is up to 30 g. of substrate per liter of broth. The fermentation is then allowed to proceed until completion is evidenced by the disappearance of steroid substrate as determined by chromatographic analysis. At the completion of the conversion, the broth is filtered and extracted with a water-immiscible solvent in which the oxygenated steroid reaction products are soluble. Suitable solvents for this purpose are, for example, halogenated hydrocarbons, e.g. methylene chloride, acetylene tetrachloride; organic acid esters, e.g. tertiary butyl acetate; aromatic hydrocarbons, e.g. benzene, toluene; ketones, diethyl ketone, cyclic amines, e.g. 2-methyl-5-ethyl-pyridine. The preferred solvents are chloroform and ethyl acetate. The solvent solution containing the product steroid is then evaporated to yield the desired product which can be further purified by recrystallization from acetone, chloroform or other standard solvent media, or subject to conventional column chromatography and recrystallization particularly in those instances, for example, where less preferred members of the genus Glomerella are employed or ll-desoxy steroids more resistant to lla-hydroxylation i.e. 1lfl-16p-alkyl steroids, are subject to the action of Glomerella. This purification procedure involves chromatographing the steroid extract obtained from the solvent solution, after evaporation, over aluminum oxide, Florisil (magnesium silicate) or like material the ratio by weight of steroid extract to, for example, magnesium silicate being normally in the ratio of 1:20 and up to 1:100 respectively. The magnesium silicate or like substance is prepared with hexane. The steroid extract is dissolved in methylene chloride for placement in the column, and the column eluted successively with a plurality of fractions, of methylene chloride, 0.5% methanol in methylene chloride, 1% methanol in methylene chloride, 1.5% methanol in methylene chloride and further increments of methanol in methylene chloride wherein the percentage of methanol is increased sequentially 0.5% in each instance until the column is eluted with 3.0% methanol and where indicated up to 5.0% methanol in methylene chloride. Elution with each fraction is normally repeated from to times with no further oil or steroid extracted. Oils are removed in the methylene chloride fraction.

When present, unconverted ll-desoxy steroid substrate is removed in the methylene chloride fraction. Any 65- hydroxylated steroid present with the lla-hydroxylated product is removed in an intermediate second fraction, and the pure lla-hydroxylated steroid is recovered in the third fraction. The pure fractions are identified by infrared comparison with authentic samples.

The process employed to identify the steroid extract for analytical purposes and to determine completion of the bio-oxidative reaction is paper chromatography. The particular paper chromatographic technique employed and that referred to throughout this specification unless otherwise explicitly indicated is that of Bush, Journal of Biochemistry, vol. 50, page 370 (1952), as modified by Shull, Paper Chromatography of Steroid Fermentation Products, 126th Meeting of the American Chemical Society, September 12 to 17, 1954, New York, New York, Section 9A, Paper No. 24.

As noted above, the lla-hydroxy steroids prepared by the process of this invention are readily converted to the corresponding ll-keto and llfl-hydroxy derivatives by known methods. Thus an illustrative procedure for oxidation of the lla-hydroxy group to the corresponding ll-keto group is described by Peterson, Eppstein et al., in the Journal of the American Chemical Society, vol. 75, pp. 412-415 (January 20, 1953), wherein, for example, 4-pregnene-11a,17a,21-triol-3,20-dione is converted to 4-pregnene-17a,2l-diol-3,11,20-trione. The conversion of 1l-keto-4-monoenes to the corresponding 11,8- hydroxy-4-monoenes is desirably accomplished by the procedure of Oliveto, Rausser et al., Journal of the American Chemical Society, vol. 78, pp. 1736-1738 (April 20, 1956). Similarly, llfi-hydroxy-lA-dienes are conveniently prepared from the corresponding 11-keto-1,4- dienes by the procedure of Herzog, Payne et al., Journal of the American Chemical Society, vol. 77, pp. 4781-4784 (September 20, 1955).

The following examples are further illustrative of the invention:

EXAMPLE 1 Agar slants containing medium No. 1 described above and 1.5% by weight of agar are sterilized for 15 minutes at 121 C. at a pressure of 15 pounds per square inch (psi). The agar slants are then cooled to about 28 C., slanted and inoculated with a vegetative growth of a culture, Glomerella cingulata (ATCC 10534) and incubated at a temperature of 28 C. until heavy sporulation occurs.

A two liter Erlenmeyer flask containing 500 milliliters (ml) of a similarly sterilized and cooled broth of medium No. 1 is then inoculated with spores from one of the heavily sporulated agar slants and incubated from 24 hours to 36 hours at 28 C. on a New Brunswick rotary shaker (manufactured by the New Brunswick Scientific Company, New Brunswick, New Jersey) set at 280 revolutions per minute (r.p.m.). At the end of this period, 500 mg. of 4-pregnene-17a,21-diol-3,20-dione in 5 m1. of ethanol is added. The flask is replaced on the rotary shaker and incubation continued for a period of from 24 hours to 30 hours during which latter period 10 ml. samples removed at intervals from the Erlenmeyer flask and extracted with chloroform are then chromatographed on paper (according to the method of Bush as modified by Shull) employing a toluene/ethyl acetate solvent system, the paper being impregnated with water and acetone, the latter being present as a dispersant which evaporates rapidly. Incubation is continued until chromatography indicates complete transformation of 4-pregnene-17a,2ldiol-3,20-dione to 4-pregnene-l1a,17a,21-triol-3,20-dione. The broth mixture in the Erlenmeyer flask is then extracted with chloroform to effect the isolation of the 4- pregnene-lla,l7a,2l-triol-3,20-dione from the broth mixture. The chloroform is then evaporated olf in vacuo and the residue is recrystallized from acetone whereby 460 mg. of 4-pregnene-11ot,17a,2l-triol-3,20-dione, melting point (M.P.) 212 C.-2l4 C., identical in all respects with an authentic sample, is obtained.

EXAMPLE 2 4-pregnene-I1 a,1 70;,21 -tri0l-3 ,2 O-dione Agar slants containing medium No. 2 described above and 1.5% by Weight of agar are sterilized for 15 minutes at 121 C. at a pressure of 15 pounds per square inch (p.s.i.). The agar slants are then cooled to about 28 C. slanted and inoculated with a vegetative growth of a culture, Glomerella cingulata (ATCC 10534) and incubated at a temperature of 28 C. until heavy sporulation occurs.

A two liter Erlenmeyer flask containing 500 milliliters (ml.) of a similarly sterilized and cooled broth of medium No. 2 is then inoculated with spores from one of the heavily sporulated agar slants and 10 ml. of anti-form, Larex (1% octadecanol in lard oil) added to the culture medium, and incubated from 24 hours to 36 hours at 28 C. on a New Brunswick rotary shaker set at 280 r.p.m. At the end of this period 500 mg. of 4-prcgnene- 17a,2l-diol-3,20dione in 5 ml. of ethanol is added. The flask is replaced on the rotary shaker and incubation continued for a period of from 24 hours to 30 hours during which latter period 10 ml. samples removed at intervals from the Erlenmeyer flask and extracted with chloroform are then chromatographed on paper (according to the method of Bush as modified by Shull) employing a 1 I toluene/ethyl acetate solvent system, the paper being impregnated with water, which is dispersed with a solvent as in Example 1. Incubation is continued until chromatography indicates complete transformation of 4-pregnene-l7nt,21-diol-3,20-dione to 4-pregnene-11u,17a,21- triol-3,20-dione. The broth mixture in the Erlenmeyer flask is then extracted with chloroform to effect the isolation of the 4-pregnene-l1a,17u,2l-triol-3,20-dione from the broth mixture. The chloroform is then evaporated off in vacuo and the residue is recrystallized from acetone.

EXAMPLE 3 4-pregnene-l I t,1 7a,21 -triol-3,20-dione Agar slants containing medium No. 3 described above and 1.5% agar are sterilized for 15 minutes at 121 C. at a pressure of 15 pounds per square inch (p.s.i.). The agar slants are then cooled to about 28C., slanted, inoculated with a vegetative growth of a culture, Glomerella lagenarium (Baarn) and incubated at a temperature of 28 C. until heavy sporulation occurs.

A two liter Erlenmeyer flask containing 500 ml. of a similarly sterilized and cooled broth of medium No. 3 is then inoculated with spores from one of the heavily sporulated agar slants and incubated for a period of from 24 hours to 36 hours at 28 C. on a New Brunswick rotary shaker set at 280 r.p.m. At the end of this period 500 mg. of 4-pregnene-l7at,21-diol-3,ZO-dione in 5 ml. of ethanol is added. The flask is replaced on the rotary shaker and incubation continued for a period of from 24 hours to 30 hours during which latter period ml. samples removed at intervals from the Erlenmeyer flask and extracted with chloroform are then chromatographed on paper (according to the method of Bush modified by Shull) employing a toluene/ethyl acetate solvent system, the paper being impregnated with water. Incubation is continued until chromatography indicates complete transformation of 4-pregncne-17a,21-diol-3,20-dione. The product is extracted from the broth mixture with chloroform. The chloroform is then evaporated off in vacuo to yield a hydroxylated extract. Twenty grams of this extract is chromatographed over 500 g. of Florisil (magnesium silicate) prepared with hexane. The steroid extract is then dissolved in methylene chloride for placement on the column, and the column eluted successively with methylene chloride, 0.5% methanol in methylene chloride, 1% methanol in methylene chloride, 1.5 methanol in methylene chloride, each elution with each of these compositions being repeated, as necessary, until no further steroid is removed in a particular fraction. Increments of 0.5% methanol in methylene chloride are made up to and including elution with 5.0% methanol in methylene chloride. Oils are removed in the methylene chloride fraction. Unconverted 4-pregnene-17a,2l-diol- 3,20-dione is also removed in the methylene chloride fraction, 4-pregnene-lla,l7u,2l-triol-3,20-dione is recovered in the 2.5% and 30% methanol containing fractions and is recrystallized from acetone.

EXAMPLE 4 4-pregneneJ1aJ 7a,2I-tri0l-3,20-di0ne The procedure of Example 3 is followed exce t that Glomerella fusarioides (ATCC 9552) was substituted for Giomerella lagermrium (Baarn). The same product, 4- pregnene-lla,17,2l-t1'iol-3,20-dione, MP. 212 C-2l4 C. is obtained.

EXAMPLE 5 I -4-pregnadiene-1 I a,I 711,21 -triol-3,20-dione Agar slants containing medium No. 3 described above and 1.5 agar are sterilized for minutes at 121 C. at a pressure of 15 pounds p.s.i. square inch (p.s.i.). The agar slats are then cooled to about 28 C. slanted and inoculated with a vegetative growth of a culture, Glamerella l'agcnarium (Baal-n) and incubated at a temperature of 28 C. until heavy sporulation occurs.

A two liter Erlenmeyer flask containing 500 ml. of a similarly sterilized and cooled broth of medium No. 3 is then inoculated with spores from one of the heavily sporulated agar slants and incubated from 24 hours to 36 hours at 28 C. on a New Brunswick rotary shaker set at 280 r.p.m. At the end of this period 500 mg. of 1,4- pregnadiene-l7a,2l-diol-3,20-dione in 5 ml. of ethanol is added. The flask is replaced on the rotary shaker and incubation continued for a period of from 24 hours to 30 hours during which latter period 10 ml. samples removed at intervals from the Erlenmeyer flask and extracted with chloroform are then chromatographed on paper (according to the method of Bush modified by Shull) as noted above employing a toluene/ethyl acetate solvent system, the paper being impregnated with water. Incubation is continued until chromatography indicates complete transformation of 1,4-pregnadiene-l7a,2l-diol- 3,20dione to 1,4-pregnadiene-11a,17a,21-triol-3,20-dione. The product is extracted from the broth mixture with chloroform. The chloroform is then evaporated oil in vacuo, subjected to column chromatography in a manner similar to that described in Example 3 and the 1,4-pregnadiene-l1a,17u,21-triol-3,20-dione recovered and recrystallized from acetone.

EXAMPLE 6 I,4-pregnadiene-1Ia,17a.2I-tri0l-3,2U-di0ne The procedure of Example 5 was followed except that Glomerella cingulata (ATCC 10529) was substituted for Glomerella lagenarium (Baarn). The result was the same as in Example 5, 1,4-pregnadiene-11a,17a,21-trio1-3,20- dione being obtained.

EXAM PLE 7 I ,4-pregnadiene-1 141,1 711,2] -tri0l-3,20-dione Agar slants containing medium No. 3 described above and 1.5% agar are sterilized for 15 minutes at 121 C. at a pressure of 15 pounds per square inch (p.s.i.). The agar slant is then cooled to about 28 C. slanted and inoculated with a vegetative growth of a culture, Glomerella cingulata (ATCC 10534) and incubated at a temperature of 28 C. until heavy sporulation occurs.

A two liter Erlenmeyer flask containing 500 ml. of a similarly sterilized and cooled broth of medium No. 3 is then inoculated with spores from one of the heavily sporulated agar slants and incubated from 24 hours to 36 hours at 28 C. on a New Brunswick rotary shaker set at 280 r.p.rn. At the end of this period 500 mg. of l.4-pregnadiene-l7n,2l-diol-3,20-dione 21- acetate in 5 ml. of ethanol is added. The flask is replaced on the rotary shaker and incubation continued for a period of from 24 hours to 30 hours during which latter period 10 ml. samples removed at intervals from the Erlenmeyer flask and extracted with chloroform are then chromatographed on paper (according to the method of Bush as modified by Shull) as noted above employing a toluene/ethyl acetate solvent system, the paper being impregnated with water. Incubation is continued until chromatography indicates complete transformation of 1,4-pregnadiene-l7a,2l-diol-3,20-dione 21- acetate to 1,4-pregnadiened1a,17e,21-triol3.20dione and the product is extracted from the broth mixture with chloroform. The chloroform is then evaporated oil in vacuo and the residue is recrystallized from acetone.

EXAMPLE 8 4-pregnene-I I a,] 701,21 -!riol-3,20-di0ne Agar slants containing medium No. 1 described above and 1.5% agar are sterilized for 15 minutes and at 121 C. at a pressure of 15 pounds psi. The agar slants are then cooled to about 28 C. slanted and inoculated with a vegetative growth of a culture Glomerella cingulata (ATCC 10531) and incubated at a temperature of 28 C. until heavy sporulation occurs.

A two liter Erlenmeyer flask containing 500 ml. of a similarly sterilized and CO.1 l6d broth of medium No. 1 is then inoculated with spores from one of the heavily sporulated agar slants and incubated from 24 hours to 36 hours at 28 C. on a New Brunswick rotary shaker set at 280 r.p.m. At the end of this period 500 mg. of 4-pregnene-17u,21-diol 3,20-dione in 5 ml. of ethanol is added. The flask is replaced on the rotary shaker and incubation continued for a period of from 24 hours to 30 hours during which latter period ml. samples removed at intervals from the Erlenmeyer flask and extracted with chloroform are then chromatographed on paper (according to the method of Bush modified by Shull) as noted above employing a toluene/ ethyl acetate solvent system, the paper being impregnated with water and acetone, the latter acting as a dispersant and evaporating. Incubation is continued until chromatography indicates complete transformation of 4-pregnene-l7e,21- diol-3,20-dione to 4 pregnene 11a,17a,21 triol 3,20- dione. The broth mixture in the Erlenmeyer flask is than extracted with chloroform to effect the isolation of the ll-hydroxylated steroid from the broth mixture. The chloroform is then evaporated off in vacuo and the residue is recrystallized from acetone to yield 4-pregnene-l1a,l7a,21-triol-3,20-dione, after being chromatographed and eluted from a column as described in Example 3.

EXAMPLE 9 Agar slants containing medium No. 1 described above and 1.5% agar are sterilized for 15 minutes at 121 C. at a pressure of 15 pounds p.s.i. The agar slants are then cooled to about 28 C. slanted and inoculated with a vegetative growth of a culture, Glomerella cingulaza (ATCC 10532) and incubated at a temperature of 28 C. until heavy sporulation occurs.

A two liter Erlenmeyer flask containing 500 ml. of a similarly sterilized and cooled broth of medium No. 1 is then inoculated with spores from one of the heavily sporulated agar slants and incubated from 24 hours to 36 hours at 28 C. on a New Brunswick rotary shaker set at 280 r. p. m. At the end of this period 500 mg. of 4-pregnene17a,2l-diol-3,20-dione in 5 m1. of ethanol is added. The flask is replaced on the rotary shaker and incubation continued for a period of from 24 to 30 hours during which latter period 10 ml. samples removed at intervals from the Erlenmeyer flask and extracted with chloroform are then chromatographed on paper (according to the method of Bush modified by Shull) employing a toluene/ ethyl solvent system, the paper being impregnated with acetone and water. Incubation is continued until chromatography indicates complete transformation of 4-pregnene-17e,2l-diol-3,20- dione to 4-pregnene-11a,]7u-2l-triol-3,20-dione. The broth mixture in the Erlenmeyer flask is then extracted with chloroform to effect the isolation of the ll-hydroxylated steroid from the broth mixture. The chloroform is then evaporated off in vacuo and the residue is recrystallized from acetone to yield 4-pregnene- 11a,17a,21-triol-3,20-dione, after being chromatographed and eluted from a column as described in Example 3.

EXAMPLE 10 4-pregnene-I1a,l 7u,21-tri0I'3,20-di0ne Agar slants containing medium No. 1 described above and 1.5% agar are sterilized for 15 minutes at 121 C. at a pressure of 15 pounds p.s.i. The agar slants are then cooled to about 28 C. slanted and inoculated with a vegetative growth of a culture, Glomerella major (Baarn) and incubated at a temperature of 28 C. until heavy sporulation occurs.

A two liter Erlenmeyer flask containing 500 m1. of a similarly sterilized and cooled broth of medium No. 1 is then inoculated with spores from one of the heavily sporulated agar slants and incubated from 24 hours to 36 hours at 28 C. on a New Brunswick rotary shaker set at 280 r.p.m. At the end of this period 500 mg. of 4-pregnene-l7e,2l-diol-3,2(]-dione in 5 ml. of ethanol is added. The flask is replaced on the rotary shaker and incubation continued for a period of from 24 hours to 30 hours during which latter period 10 ml. samples removed at intervals from the Erlenmeyer flask and extracted with chloroform are then chromatographed on paper (according to the method of Bush as modified by Shull) employing a toluene/ethyl acetate solvent system, the paper being impregnated with acetone and water. Incubation is continued until chromatography indicates complete transformation of 4-pregnene-17a,2ldiol-3 ,ZO-dione to 4-pregnene- 1 101,1 7a,2 1-triol-3,20-dione. The broth mixture in the Erlenmeyer flask is then extracted with chloroform to effect the isolation of the llhydroxylated steroid from the broth mixture. The chloroform is then evaporated off in vacuo and the residue is recrystallized from acetone to yield 4-pregnene- 1lot,i7oz,2l-U10l-3,20di0l16, after being chromatographed and eluted from a column as described in Example 3.

EXAMPLE ll 4-pregnene-11 m17a,21-triol-3,20-di0ne Agar slants containing medium No. 1 described above and 1.5% agar are sterilized for 15 minutes at 121 C. at a pressure of 15 pounds per square inch (p.s.i.). The agar slants are then cooled to about 28 C. slanted and inoculated with a vegetative growth of a culture, Glamerella phacidiomorpha (Baarn) and incubated at a temperature of 28 C. until heavy sporulation occurs.

A two liter Erlenmeyer flask containing 500 ml. of a similarly sterilized and cooled broth of medium No. 1 is then inoculated with spores from one of the heavily sporulated agar slants and incubated from 24 hours to 36 hours at 28 C. on a New Brunswick rotary shaker (manufactured by the New Brunswick Scientific Company, New Brunswick, New Jersey) set at 280 r.p.m. At the end of this period 500 mg. of 4-pregnene-l7a,21-diol-3,20dione in 5 ml. of ethanol is added. The flask is replaced on the rotary shaker and incubation continued for a period of from 24 hours to 30 hours during which latter period 10 ml. samples removed at intervals from the Erlenmeyer flask and extracted with chloroform are then chromatographed on paper (according to the method of Bush as modified by Shull) employing a toluene/ ethyl acetate solvent system, the paper being impregnated with acetone and water. Incubation is continued until chromatography indicates complete transformation of 4-pregnene-l7a,2ldiol-3 ,20-dione to 4-pregnene-l la, 17 01,2 1 -trio1-3 ,20-dione. The broth mixture in the Erlenmeyer flask is then extracted with chloroform to effect the isolation of the 11- hydroxylated steroid from the broth mixture. The chloroform is then evaporated off in vacuo and the residue recrystallized from acetone to yield 4-pregnene-11u,l7a,2ltriol-3,20-dione, after being chromatographed and eluted from a column as described in Example 3.

EXAMPLE l2 4-pregnene-I I a,I7ot,21-triol-3,20-di0ne Agar slants containing medium No. 1 described above and 1.5% agar are sterilized for 15 minutes at 121 C. at a pressure of 15 pounds per square inch (psi). The agar slants are then cooled to about 28 C. slanted and inoculated with a vegetative growth of a culture, Glomerella rubicola (Baaran) and incubated at a temperature of 28 C. until heavy sporulation occurs.

A two liter Erlenmeyer flask containing 500 milliliters (ml.) of a similarly sterilized and cooled broth of medium No. 1 is then inoculated with spores from one of the heavily sporulated agar slants and incubated from 24 hours to 36 hours at 28 C. on a New Brunswick rotary shaker (manufactured by the New Brunswick Scientific Company, New Brunswick, New Jersey) set at 280 r.p.m.

At the end of this period 500 mg. of 4p-regnene-17u,21- diol-3,20-dione in 5 ml. of ethanol is added. The flask is replaced on the rotary shaker and incubation continued for a period of from 24 hours to 30 hours during which latter period 10 ml. samples removed at intervals from the Erlenmeyer flask and extracted with chloroform are then chromatographed on paper (according to the method of Bush modified by Shull) employing a toluene/ ethyl acetate solvent system, the paper being impregnated with acetone and water. Incubation is continued until chromatography indicates complete transformation of 4 pregnenel7a,21-diol-3,20-di0ne to 4-pregnenellm,17o=, 2l-triol-3,20-dione. The broth mixture in the Erlenmeyer flask is then extracted with chloroform to eflect the isolation of the ll-hydroxylated steroid from the broth mixture. The chloroform is then evaporated in vacuo and the residue is recrystallized from acetone to yield 4-pregnene-l1a,17a,21-triol-3,20-dione after being chromatographed and eluted from a column as described in Example 3.

EXAMPLE 13 4-pregnene-I 1a,] 701,21 -trioI-3,20-din2 Agar slants containing medium No. 1 described above and 1.5 agar are sterilized for 15 minutes at 121 C. at a pressure of 15 pounds per square inch (psi). The agar slants are then cooled to about 28 C. slanted and inoculated with a vegetative growth of a culture, Glomerella cingulam (ATCC l0530) and incubated at a temperature of 28 C. until heavy sporulation occurs.

A two liter Erlenmeyer flask containing 500 ml. of a similarly sterilized and cooled broth of medium No. 1 is then inoculated with spores from one of the heavily sporulated agar slants and incubated from 24 hours to 36 hours at 28 C. on a New Brunswick rotary shaker (manufactured by the New Brunswick Scientific Company, New Brunswick, New Jersey) set at 280 r.p.m. At the end of this period 500 mg. of 4-pregnene-l7u,2l-diol- 3,20-dione in 5 ml. of ethanol is added. The flask is replaced on the rotary shaker and incubation continued for a period of from 24 hours to 30 hours during which latter period ml. samples removed at intervals from the Erlenmeyer flask and extracted with chloroform. are then chromatographed on paper (according to the method of Bush as modified by Shull) employing a toluene/ethyl acetate solvent system, the paper being impregnated with acetone and water. Incubation is continued until chromatography indicates complete transformation of 4-pregnene-l7a,2l-diol-3,20-dione to 4 pregnene l1oL,l7a,2ltriol-3.20-dione. The broth mixture in the Erlenmeyer flask is then extracted with chloroform to effect the isolation of the ll-hydroxylated steroid from the broth mixture. The chloroform is then evaporated in vacuo and the residue is recrystallized from acetone to yield 4-pregnene-lla,l7e,2l-triol-3,20-dione, after being chromatograph-ed and eluted from a column as described in Example 3.

EXAMPLE 14 I 6a-methylallopregnane-l I 11,] 7 ot,2I -tri0l-3,20 di0ne (a) 1 6a-METHYLALLOPREGNAN-3B-OL-QO-ONE A solution of 0.5 g. l6-a-methylpregnenolone in 15 ml. of acetic acid is reduced at atmospheric pressure with hydrogen and 0.3 g. of 5% palladium on charcoal catalyst. The reaction is stopped after one mole of hydrogen is consumed, the catalyst is removed by filtration, and the filtrate poured into water. The precipitated solid is removed by filtration and crystallized from acetonehexane to yield 0.3 g. of l6a-methylallopregnan-3 3-ol-20-one.

(b) 16a-METHYLALLOPREGNANE-Sfi,17a-DIOL-20-ONE A solution of 3.5 g. of lfiwmethylallopregnan-3 3-01-20- one in 100 ml. of acetic anhydride containing 2.0 g. of ptoluene-sulfonic acid is kept at l00 C. for 6 hours; during this time about 8 ml. of distillate is removed each half hour by the application of vacuum. The resulting oily residue is dissolved in 50 ml. of benzene and washed three times with water; then with a solution of 1.0 g. of sodium acetate in 15 ml. of water. The benzene layer is then stirred for 18 hours at room temperature with a mixture of 0.25 g. of sodium acetate in 6 ml. of commercial 40% peracetic acid. The excess peracetic acid is destroyed by the dropwise addition of a solution of 8 g. of sodium sulfite in 25 ml. of water, at a temperature of ca. 10-20". An additional 1 g. of sodium sulfite is then added and the mixture stirred overnight until a starch-iodide test is negative. The benzene layer is separated, washed three times with water and evaporated. The residue is dissolved in 200 ml. of methanol and 30 ml. of water containing 2.7 g. of sodium hydroxide, and the mixture refluxed for 15 minutes. After neutralization with 3 ml. of acetic acid, the solution is concentrated under reduced pressure to a volume of ca. 30 ml., and this is poured into an ice-water mixture. The precipitated solid is removed by filtration and crystallized from methanol-Water to give 2.5 g. of lGa-methylallopregnane-3B, l7u-diol-20-dio-ne.

(c) 1Ba-METHYLALLOPREGNANE-liflJ7a,21-TRIOL- 20-0Nn ZI-ACETATE A solution of 300 mg. of 16a-methylallopregnane-3fi, l7a-diol-20-one in 15 ml. of C.P. chloroform (containing a few drops of chloroform previously saturated with hydrogen bromide) is brominated at 20 by the addition (over a two hour period) of 165 mg. of bromine in 10 ml. of chloroform. After removal of the solvent under re duced pressure, 10 ml. of dimethylformamide and one gram of sodium acetate are added. The mixture is stirred at 60-70 for 16 hours, then poured into water and the precipitated solid removed by filtration. Crystallization from acetone-hexane to give 200 mg. of Mot-methylallopregnane-Iifl, 1 704,2 1 -triol-20-one 2 l -acetate.

a 1Ga-METHYLALLOPREGNANE-l7a,21-DIOL-3,20- DIONE 21-ACETATE A solution of 3.5 g. of l6a-methylallopregnan3fl,17a, 21-triol-20-one in 20 ml. of acetone-water is cooled to 10 C. One drop of concentrated hydrochloric acid is added along with 200 mg. of N-bromoacetamide, and the mixture allowed to stand in the ice-box for 20 hours. Excess sodium sulfite solution is then added, and the mixture concentrated under reduced pressure to a small volume to precipitate a crude product. This is crystallized from acetone-hexane to yield 3.0 g. of l6a-methylallopregnane-l7a,2l-diol-3,20-dione 2l-acetate.

(e) 1Ba-METHYLALLOPREGNANE-l7a,21-DIOL- 3,20-DIONE One gram of Ida-methylallopregnane-l7a,2l-diol-3,20- dione 21-acetate is dissolved in 25 ml. of methanol and 5 ml. of water containing 0.2 g. of potassium bicarbonate. This solution is refluxed for /2 hour, then concentrated under reduced pressure. Water is added to the residue, and the resulting precipitate is filtered and dried. Crystallization from acetone-hexane gives l6a-rnethylallopregnane-l7a,2l-diol-3,20-dione.

(f) 1(la-METHYLALLOPREGNANE-llo,17a,21-TRIOL- 3,20-DIONE Agar slants containing medium No. 1 described above and 1.5% agar are sterilized for 15 minutes and at 121" C. at a pressure of 15 pounds p.s.i. The agar slants are then cooled to about 28 C. slanted and inoculated with a vegetative growth of a culture Glomerella cingulata (ATCC 10534) and incubated at a temperature of 28 C. until heavy sporulation occurs.

A two liter Erlenmeyer flask containing 500 ml. of a similarly sterilized and cooled broth of medium No. 1 is then inoculated with spores from one of the heavily sporulated agar slants and incubated from 24 hours to 36 hours at 28 C. on a New Brunswick rotary shaker set at 280 r.p.m. At the end of this period 500 mg. of mot-methylallopregnane-17a.21-diol-3,20-dione in 5 ml. of ethanol is added. The flask is replaced on the rotary shaker and incubation continued for a period of from 24 hours to 30 hours during which latter period ml. samples removed at intervals from the Erlenmeyer flask and extracted with chloroform are then chromatographed on paper (according to the method of Bush as modified by Shull) employing a toluene/ethyl acetate solvent system, the paper being impregnated with acetone and water. Incubation is continued until chromatography indicates complete transformation of la-methylallopgregnanel7a,2l-diol-3, ZO-dione to 16a-methylallopregnane-l la,17a,2l-t1'iOl-3, 20-dione. The broth mixture in the Erlenmeyer flask is then extracted with chloroform to cfiect the isolation of the ll-hydroxylated steroid from the broth mixture. The chloroform is then evaporated oil in vacuo and the residue is recrystallized from acetone to yield l6a-methylallopregnane-l1a,l7u,2l-triol-3,20-dione, after being chromatographed and eluted from a column as described in Example 3.

EXAMPLE l5 I6B-methylallopregnane-11a,1 7a,21-tri0l-3,20-di0ne (a) 16B METHYLALLOPREGNAN-3fi-OL-20-ONE In the manner described in Example 14(a), 1613- methylpregnenolone is reduced to IGB-methylallopreg nan-Bfi-ol-ZO-one by means of hydrogen and a palladium catalyst.

(b) l6BJMETHYLALLOPREGNANE-3fi,17a-DIOL-20-ONE In the manner described in Example 14(b) l6 3-methylallopregnan-Zifl-ol-ZO-one is enol-acetylated, peroxidized and hydrolyzed to give 16fl-methylallopregnane-3fl,17adiol-20-one.

(e) 1GB-METHYLALLOPREGNANE-BBJ711,21-TRIOL- 20-ONE tel-ACETATE In the manner described in Example 14(0), lfl-methylallopregnane-3fl,17e-diol-20-one is brominated and acetoxylated at C-2l to give l6,6-methylallopregnane-33J7a, 21-trio1-20-one 2 l -acetate.

(d) 1 GB-METHYLALLOPREGNANE-l 7a,21-DIOL-3,20-

DIONE 21-ACETATE In the manner described in Example 14(d), IGB-methylallopregnane-3fi,17a,2l-triol-ZO-one 21-acetate is oxidized to give 1Gtt-methylallopregnane-17e,2l-diol-3,20-dione 21- acetate.

(8) 1BB-METHYLALLOPREGNANE-l7a,21-DIOL- 3,20-DIONE One gram of l6B-methylallopregnane-170:,2l-diol-3,2O- dione 21-acetate is dissolved in 25 ml. of methanol and 5 ml. of water containing 0.2 g. of potassium bicarbonate. This solution is refluxed for V2 hour, then concentrated under reduced pressure. Water is added to the residue, and the resulting precipitate is filtered and dried. Crystallization from acetone-hexane gives l6fl-methylallopregnane-l 1a,21-diol-3,20-dione.

(f) 1 Gfl-METHYLALLOPREGNANE-l la,1 Ta-ZI-TRIOL- 3,20-DIONE Agar slants containing medium No. 1 described above and 1.5% agar are sterilized for minutes and at 121 C. at a pressure of 15 p.s.i. The agar slants are then cooled to about 28 C. slanted and inoculated with a vegetative growth of a culture Glomerella cingulata (ATCC 10534) and incubated at a temperature of 28 C. until heavy sporulation occurs.

A two liter Erlenmeyer flask containing 500 ml. of a similarly sterilized and cooled broth of medium No. 1 is then inoculated with spores from one of the heavily sporulated agar slants and incubated from 24 hours to 36 hours at 28 C. on a New Brunswick rotary shaker set at 280 r.p.m. At the end of this period 500 mg. of 16B-methylallopregnane-l7e,2l-diol-3,20-dione in 5 ml. of ethanol is added. The flask is replaced on the rotary shaker and incubation continued for a period of from 24 hours to 30 hours during which latter period 10 ml. samples removed at intervals from the Erlenmeyer flask and extracted with chloroform are then chromatographed on paper (according to the method of Bush as modified by Shull) employing a toluene/ethyl acetate solvent system, the paper being impregnated with acetone and water. Incubation is continued until chromatography indicates complete transformation of l6fi-methylallopregnane-l7a,2ldiol-3,20-dione to 16B-methylallopregnane-1le,17a,21- triol-3,20-dione. The broth mixture in the Erlenmeyer flask is then extracted with chloroform to eflect the isolation of the ll-hydroxylated steroid from the broth mixture. The chloroform is then evaporated oiT in vacuo and the residue is recrystallized from acetone to yield 166- methylallopregnane-l 1,l7a,21-triol-3,20-dione.

EXAMPLE 16 16a-tart.-butylallopregnane-lI(1,17a,21-triol-3,20-dione (a) 16a-TE-RT.-BUTYLALLOPREGNAN-Bfl-OIrZO-ONE In the manner described in Example 14(11), Ida-ten.- butylpregnenolone is reduced to loa-tert-butylallopregnan-Bfl-ol-ZO-one by means of hydrogen and a palladium catalyst.

(b) l.6a-TERT.-BUTYLALLOPREGNANE-3fl,17a-DIOL- 20-ONE In the manner described in Example 14(b), l6a-tert.- butylallopregnan-Bfi-ol-20 one is enol acetylated, peroxidized and hydrolyzed to give l6u-tert.-butylallopregnane-3fi,17a-diol-20-one.

(c) 16wTERT.-BUTYLALLOPREGNANE-3B,17a,21TRIOL 20-0NE ZI-ACETATE In the manner described in Example 14(c), l6e-tert.- butylallopregnane-Zifl,l7a-diol-20-one is brominated and acetoxylated at C-2l to give l6a-tert.-butylallopregnane- 3;3,l7a,21-triol-20-one 21-acetate.

(d) 1fia-TERT.-BUTYLALLOPREGNANEA7a,21-DIOL-3,20- DIONE 21-ACE'1ATE In the manner described in Example 14(d), l6a-tert.- butylallopregnane-3B,17e,21-trio]-20-one 21-acetate is oxidized to give l6a-tert.-butylallopregnane-17a,2l-diol- 3,20-dione ZI-acetate.

(e) 1.6n-TERT.-BUTYLALLOPBEGNANE-17a,21-DIOL- 3,20-DIONE One gram of 16a-tert.-butylallopregnane-1711,2l-diol- 3,20-dione 2l-acetate is dissolved in 25 ml. of methanol and 5 ml. of water containing 0.2 g. of potassium bicarbonate. This solution is refluxed for /2 hour, then concentrated under reduced pressure. Water is added to the residue, and the resulting precipitate is filtered and dried. Crystallization from acetone-hexane gives 16atert.-butylallopregnane-l7a,2l-diol-3,20-dione.

( f) 1fia-TERT.-BUTYLALLOPREGNANE-11a,17a,21- TRIOL3,20DIONE Agar slants containing medium No. 1 described above and 1.5% agar are sterilized for 15 minutes and at 121 C. at a pressure of 15 p.s.i. The agar slants are then cooled to about 28 C. slanted and inoculated with a vegetative growth of a culture Glomerelia cingulata (A'ICC 10534) and incubated at a temperature of 28 C. until heavy sporulation occurs.

A two liter Erlenmeyer flask containing 500 ml. of a similarly sterilized and cooled broth of medium No. l is then inoculated with spores from one of the heavily sporulated agar slants and incubated from 24 hours to 36 hours at 28 C. on a New Brunswick rotary shaker set at 280 r.p.m. At the end of this period 500 mg. of lGot-text.-butylallopregnane-l7a,2l-diol-3,20-dione in 5 ml. of ethanol is added. The flask is replaced on the rotary shaker and incubation continued for a period of from 24 hours to 30 hours during which latter period 10 ml. samples removed at intervals from the Erlenmeyer flask and extracted with chloroform are then chromatographed on paper (according to the method of Bush as modified by Shull) employing a toluene/ethyl acetate solvent system, the paper being impregnated with acetone and water. Incubation is continued until chromatography indicates complete transformation of 16a-tert.-butylallopregnane-l7a,21-diol-3,20-dione to 16a-terL-butylallopregnane-l loz,l7ot,21-lt'iO1-3,2U-di0t't. The broth mixture in the Erlenmeyer flask is then extracted with chloroform to effect the isolation of the ll-hydroxylated steroid from the broth mixture. The chloroform is then evaporated off in vacuo and the residue is recrystallized from acetone to yield 16a-tert.-butylallopregnane-lla,17m,2ltriol-3,20-dione, after being chromatographed and eluted from a column as described in Example 3.

EXAMPLE 17 165-ethylalIopregnane-1 1 a,] 70:,21 -tri0I-3,20-dione (a) 16B-ETHYLALLOPREGNANE-iiB-OLQO-ONE A solution of 3.0 g. of l6-dehydropregnenolone in 6 ml. of methylene chloride is added to a solution of about 1 g. of diazoethane in 50 ml. of ether at about 0 C. The mixture is kept at this temperature for 6 hours, then allowed to warm to room temperature. Removal of the solvent leaves a residue of the intermediate pyrazoline, which is not further purified, but heated under reduced pressure to ca. 200 C. until the evolution of nitrogen ceases. The resulting oil is crystallized from ether to give 2.0 g. of 16-ethyl-ltS-dehydropregnenolone. This is dissolved in 50 ml. of acetic acid, then reduced with hydrogen and a palladium on charcoal catalyst until 2 moles of hydrogen have been absorbed. The catalyst is removed by filtration, and the filtrate poured into water. The precipitated solid is removed by filtration and crystallized from methanol-water to yield 1.5 g. of 165- ethylallopregnan-IiB-ol-ZO-one.

In the manner described in Example 14(1)). loo-ethylallopregnane-Sfi-ol-20one is enol-acetylated, peroxidized and hydrolyzed to give lBis-ethylallopregnancdfi,17adiol-ZO-one.

(c) 16fl-ETHYLALLOPREGNANE-3B,17a,21-TRIOL-20 ONE 21-ACETATE In the manner described in Example 14(c), IGfl-ethylallopregnane-Bfl,l7rx-diol-20-one is brominated and acetoxylated at C-Zl to give 1GB-ethylallopregnane-Bfl,17a, 2 l -triol-20-one 2 l -acetate.

(11) 1GB-ETHYLALLOPREGNANE-l7:1,21-DIOL-3,20-DIONE ill-ACETATE In the manner described in Example 14(d), ltSB-ethylallopregnane-3fl,l7u,2l-triol-2'0-one 2l-acctate is oxidized to give lfifi-ethylallopregnane-l7d,21-diol-3,20-dionc 21- acetate.

(e) 1fifi-ETHYLALLOPREGNANE-l'ln,21DIOL- 3,20-DIONE One gram of l6B-ethylallopregnane-l7a,2l-diol-3,20- dione 2l-acetate is dissolved in 25 ml. of methanol and ml. of water containing 0.2 g. of potassium bicarbonate. This solution is refluxed for V2 hour, then concentrated under reduced pressure. Water is added to the residue, and the resulting precipitate is filtered and dried. Crystallization from acetone-hexane gives 16B-ethylallopregnancl 7a,2 l-diol-3,20-dione.

(f) 16 ETHYLALLOPREGNANE11a,1711,21-TRIOL- 3,20-DIONE Agar slants containing medium No. 1 described above and 1.5% agar are sterilized for 15 minutes and at 121 C. at a pressure of 15 p.s.i. The agar slants are then cooled to about 28 C. slanted and inoculated with a vegetative growth of a culture Glomerelln cr'ngulata (A'l' CC 10534) and incubated at a temperature of 2t C. until heavy sporulation occurs.

A two liter Erlenmeyer flask containing 500 ml. of a similarly sterilized and cooled broth of medium No. l is then inoculated with spores from one of the heavily sporulated agar slants and incubated from 24 hours to 36 hours at 28 C. on a New Brunswick rotary shaker set at 280 rpm. At the end of this period 500 mg. of IGfi-ethylallopregnane-l7a,2l-diol-3,20-dione in 5 ml. of ethanol is added. The flask is replaced on the rotary shaker and incubation continued for a period of from 24 hours to 30 hours during which latter period 10 ml. samples removed at intervals from the Erlenmeyer flask and extracted with chloroform are then chromatographed on paper (according to the method of Bush as modified by Shull) employing a toluene/ethyl acetate solvent system, the paper being impregnated with acetone and water. Incubation is continued until chromatography indicates complete transformation of 16,3-ethylallopregnane-l7a. 2l-diol-3,20-dione to lofi-ethylallopregnane-llot,l7a,2ltriol-3,20-dione. The broth mixture in the Erlenmeyer flask is then extracted with chloroform to effect the isolation of the ll-hydroxylatcd steroid from the broth mixture. The chloroform is then evaporated off in vacuo and the residue is recrystallized from acetone to yield 1ofi-ethylallopregnaned 1a, 1 7a,2 l -triol-3,20-dionc.

EXAMPLE 18 I6u-methyl-I ,4-pregnadieneJ1 a, 17:1,21 -tri0'l- 3,20-dione (a) 16a-METHYL-1,4 PRElGNADIENE-1701,21DIOL- 3,20-DIONE 21-ACETATE A solution of 200 mg. of lou-methylallopregnane-17m, 21-diol-3,20-dione ZI-acetate obtained as described in Example l4(ad) in 5 ml. of dioxane is dibrominated in positions 2 and 4 by the rapid addition of 130 mg. of bromine in 1 ml. of dioxane at room temperature. After /2 hour, the solution is poured into water and the precipitated solid removed by filtration and dried. Without further purification, this is dehydrobrominated by refluxing for 2 hours with 4 ml. of dimethylformamide containing 50 mg. of lithium carbonate and 50 mg. of lithium bromide. The mixture is then poured into dilute hydrochloric acid and is extracted with methylene chloride. The organic extracts are evaporated to a residue which is crystallized from methanol-water to yield mg. of 1fia-methyl-l,4-pregnadiene-l7a,21-dio1-3,20-dione ZI-acetate.

(b) lfia-METHYL-l.4-PREGNAD1ENE-llaZl-DIOL- 3,20-DIONE One gram of 1fia-rnethyl-l,4-pregnadiene-I7m,2l-diol- 3,20-dione 21-acetate is dissolved in 25 ml. of methanol and 5 ml. of water containing 0.2 g. of potassium bicarbonate. This solution is refluxed for V2 hour, then concentrated under reduced pressure. Water is added to the residue, and the resulting precipitate is filtered and dried. Crystallization from acetone-hexane gives 16amethyl-1,4-pregnadicne-17a,2l-diol-3,20,-dione.

(c) 1Ga-METHYL-l,*l-PREGNADIENEJlaJ70,21- TRIOL3,20-DIONE Agar slants containing medium No. 1 described above and 1.5% agar are sterilized for 15 minutes at 121 C. at a pressure of 15 p.s.i. The agar slants are then cooled to about 28 C. slanted and inoculated with a vegetative growth of a culture Glomerella cingulata (ATCC 10534) and incubated at a temperature of 28 C. until heavy sporulation occurs.

A two liter Erlenmeyer flask containing 500 ml. of a similarly sterilized and cooled broth of medium No. l is then inoculated with spores from one of the heavily sporulated agar slants and incubated from 24 hours to 36 hours at 28" C. on a New Brunswick rotary shaker set at 280 rpm. At the end of this period 500 mg. of 1da-methyl-l,4-pregnadiene-l7u,2l-diol-3,20-dione in 5 ml. of ethanol is added. The flask is replaced on the rotary shaker and incubation continued for a period of from 24 hours to 30 hours during which latter period 10 ml. samples removed at intervals from the Erlenmeyer flask and extracted with chloroform are then chromato- EXAMPLE 19 16p-methyI-1,4-pregnadiene-I 1 01,] 704,21- trial-3,20-dione (a) IGH-METHYL-l .4-PREGNADIENE-1 7a,21-DlOL-3,2ll- DIONE 21-ACETATE In the manner described in Example 18(a), 16fl-methy1allopregnane-l7a,21 diol 3,20-dione ZI-acetate obtained by the procedure described in Example 15(a-d) is dibrominated in positions 2 and 4, then dehydrobrominated to give lfifl-methyl-l,4-pregnadiene-17u,2l-diol- 3,20-dione 2l-acetate.

(b) 16fl-METIIYL1,4-PREGNADIENE17a,21-DIOL 3.20-DIONE One gram of lofl-methyl-l,4-pregnadiene-l7a.,2l-diol- 3.20-dione 2l-acetate is dissolved in 25 ml. of methanol and 5 ml. of water containing 0.2 g. of potassium bicarbonate. This solution is refluxed for 96. hour, then concentrated under reduced pressure. Water is added to the residue, and the resulting precipitate is filtered and dried. Crystallization from acetone-hexane gives lop-methyl- ,4-pregnadiene-l7a,21-diol-3,20-dione.

[) 16B METHYL-1,4-PREGNADIENE-11a,1 70.,21-THIOL- 3,20-DIONE Agar slants containing medium No. 1 described above and 1.5% agar are sterilized for 15 minutes and at 121 C. at a pressure of 15 p.s.i. The agar slants are then cooled to about 28 C. slanted and inoculated with a vegetative growth of a culture Glamerella cingulata (ATCC 10534) and incubated at a temperature of 28 C. until heavy sporulation occurs.

A two liter Erlenmeyer flask containing 500 ml. of a similarly sterilized and cooled broth of medium No. l is then inoculated with spores from one of the heavily sporulated agar slants and incubated from 24 hours to 36 hours at 28 C. on a New Brunswick rotary shaker set at 280 r.p.m. At the end of this period 500 mg. of 16p-methyl-l,4-pregnadiene-l7a-21-diol-3,20-dione in ml. of ethanol is added. The flask is replaced on the rotary shaker and incubation continued for a period of from 24 hours to 30 hours during which latter period ml. samples removed at intervals from the Erlenmeyer flask and extracted with chloroform are then chromatographed on paper (according to the method of Bush as modified by Shull) employing a toluene/ethyl acetate solvent system, the paper being impregnated with acetone and water. Incubation is continued until chromatography indicates complete transformation of lfifl-methyl-l,4-pregnadiene-17a,21-diol-3,20dione to lp-methyl- 1,4 pregnadiene-l1a,17a,2l-triol-3,20-dione. The broth mixture in the Erlenmeyer flask is then extracted with chloroform to effect the isolation of the 11-hydroxylated steroid from the broth mixture. The chloroform is then evaporated oif in vacuo and the residue is recrystallized from acetone to yield lfifl-methyl-l,4-pregnadiene-lla, l7a,2l-triol-3,20-dione, after being ehromatographed and eluted from a column as described in Example 3.

22 EXAMPLE 20 16a-terL-butyl-1,4-pregnadiene-II 0:,1 7 u,21 trial-3,20-dl'0fle (a) 16a-TERT.-BUTYL-1,4-P1tEGNADIENEJMQLDIOL- 3,20-DIONE 21-ACE'IATE In the manner described in Example 18(0), 16a-tert.- butylallopregnane l7a,21-diol-3,20-dione ZI-acetate obtained by the procedure described in Example 16(a-d) is dibrominated in positions 2 and 4, then dehydrobrominated to give l6a-tert.-butyl-1,4-pregnadiene-l7a,2l-diol- 3,20-dione 2l-acetate.

(b) ma-TERLBUTYIA,4-P1tEGNADIENE-1Ta21-DIOL- ago-moms One gram of l6a-tert.-butyl-1,4-pregnadiene-17a,2ldiol-3,20-dione Zl-acetate is dissolved in 25 ml. of methanol and 5 ml. of water containing 0.2 g. of potassium bicarbonate. This solution is refluxed for l. hour, then concentrated under reduced pressure. Water is added to the residue, and the resulting precipitate is filtered and dried. Crystallization from acetone-hexane gives 16atert.-butyll ,4-pregnadiene-l 7a,2 l-diol-3 ,20-dione.

(c) 1timTERT. BUTYL-1,4-PREGxADIENE-11a,17a,21-

TRIOL BQO-DIONE Agar slants containing medium No. 1 described above and 1.5% agar are sterilized for 15 minutes and at 121 C. at a pressure of 15 psi. The agar slants are then cooled to about 28 C. slanted and inoculated with a vegetative growth of a culture Glomerella lagenarium (Baarn) and incubated at a temperature of 28 C. until heavy sporulation occurs.

A two liter Erlenmeyer flash containing 500 ml. of a similarly sterilized and cooled broth of medium No. 1 is then inoculated with spores from one of the heavily sporulated agar slants and incubated from 24 hours to 36 hours at 28 C. on a New Brunswick rotary shaker set at 280 rpm. At the end of this period 500 mg. of l6a-tert.- butyl-l,4-pregnadiene-l7a,2l-diol-3,20-dione in 5 ml. of ethanol is added. The flask is replaced on a rotary shaker and incubation continued for a period of from 24 hours to 30 hours during which latter period 10 ml. samples removed at intervals from the Erlenmeyer flask and extracted with chloroform are then chromatographed on paper (according to the method of Bush as modified by Shull) employing a toluene/ethyl acetate solvent system, the paper being impregnated with acetone and water. Incubation is continued until chromatography indicates complete transformation of l6a-tert.-butyl-l,4 pregnadiene-l7u,2l-diol-3,20-dione to l6a-tert.-butyl-l,4 pregnadiene-lla,17a,2l-triol-3,20dione. The broth mixture in the Erlenmeyer flask is then extracted with chloroform to eiiect the isolation of the ll-hydroxylated steroid from the broth mixture. The chloroform is then evaporated ofi in vacuo and the residue is recrystallized from acetone to yield lot-tert-butyl-l,4-pregnadiene-lla.l7a, 2l-triol-3,20-dione, after being chromatographcd and eluted from a column as described in Example 3.

EXAMPLE 2] 16,8-ethyi-1 ,4-pregnadiene-11 0a,] 7a,21-!ri0I-3,20-di0ne (rt) lfifl-ETHYL-l,4-PREGNADIENE-17a,21-DIOL-3,20- DIONE 21-ACETATE in the manner described in Example 18 (0.), l6fi-ethylallopregnane-l7u,2l-diol-3,20-dione 2l-acetate obtained by the procedure described in Example 17 (a-d) is dibrorninated in positions 2 and 4, then dehydrobrominated to give 16--B-ethyl-l,4-pregnadicne-l7a,2l-diol-3,20-dione ill-acetate.

(b) tee-ls'rrtYL-l,4-PREGNADIENE. 17o,21-DIOL-3,20-DIONE One gram of lfifi-ethyl-l,4-pregnadiene-17,2l-di0l-3, 20-dione ZI-acetate is dissolved in 25 ml. of methanol and 5 ml. of water containing 0.2 g. of potassium bicarbonate. This solution is refluxed for /2 hour, then concentrated under reduced pressure. Water is added to the residue, and the resulting precipitate is filtered and dried. Crystallization from acetone-hexane gives 16fl-ethyl-1,4- pregnadiene-l7a,2l-diol-3,20-dione.

(c) 16B-ETHYL-1,4-PREGNADIENE-11a,17a.,2l-TRIOL- 3.2o-DIONE Agar slants containing medium No. 1 described above and 1.5% agar are sterilized for 15 minutes and at 121 C. at a pressure of 15 pounds psi. The agar slants are then cooled to about 28 C. slanted and inoculated with a vegetative growth of a culture Glomerella cingulata (ATCC 10534) and incubated at a temperature of 28 C. until heavy sporulation occurs.

A two liter Erlenmeyer flask containing 500 ml. of a similarly sterilized and cooled broth of medium No. 1 is then inoculated with spores from one of the heavily sporulated agar slants and incubated from 24 hours to 36 hours at 28 C. on a New Brunswick rotary shaker set at 280 r.p.m. At the end of this period 500 mg. of 16-5- ethyl-1,4-pregnadiene-17a,2ldiol-3,20-dione in ml. of ethanol is added. The flask is replaced on the rotary shaker and incubation continued for a period of from 24 hours to 30 hours during which latter period ml. samples removed at intervals from the Erlenmeyer flask and extracted with chloroform are then chromatographed on paper (according to the method of Bush as modified by Shull) employing a toluene/ethyl acetate solvent system, the paper being impregnated with acetone and water. Incubation is continued until chromatography indicates Complete transformation of lnfi-ethyl-l,4-pregnadiene- 170:.2 l-diol-3.2(l-dione to lon-ethyl-t ,4-pregnadiene-1 la, l70:,2l-triol3,2il-di0ne. The broth mixture in the Erlenmeyer flask is then extracted with chloroform to effect the isolation of the ll-hydroxylated steroid from the broth mixture. The chloroform is then evaporated off in vacuo and the residue is recrystallized from acetone to yield 16,8- ethy'l-lA-pregnadiene lla,l7rz,2l-l1l0l-3,ZO-dl0n, after being chromatograplied ad eluted from a column as de scribed in Example 3.

EXAMPLE 22 1 da-methylaliopregnane-I 1 (1,] 711,21 -triol-3,20-dione Agar slants containing medium No. 1 described above and 1.5% by weight agar are sterilized for minutes at 121 C. at a pressure of 15 pounds p.s.i. The agar slants are then cooled to about 28 C. slanted and inoculated With a vegetative growth of a culture Glomerella singulata (ATCC 10529) and incubated at a temperature of 28 C. until heavy sporulation occurs.

A two liter Erlenmeyer flask containing 500 ml. of a similarly sterilized and cooled broth of medium No. 1 is then inoculated with spores from one of the heavily sporulated agar slants and incubated from 24 hours to 36 hours at 28 C. on a New Brunswick rotary shaker set at 280 r.p.m. At the end of this period 500 mg. of l6zt-methylallopregnane-1711,21 diol 3,20 dione, prepared as described in Example 14 (ae), in 5 ml. of ethanol is added. The flask is replaced on the rotary shaker and incubation continued for a period of from 24 hours to 30 hours during which latter period 10 ml. samples removed at intervals from the Erlenmeyer flask and extracted with chloroform are then chromatographed on paper (according to the method of Bush as modified by Shull) employing a toluene/ethyl acetate solvent system, the paper being impregnated with acetone and water. Incubation is continued until chromatography indicates complete transformation of .ltx-mcthylallopregnane-l7a, 2l-diol-3,20-dione to ltSa-methylallopregnane-lla,17a,21- triol-3,20-dione. The broth mixture in the Erlenmeyer flask is then extracted with chloroform to elfect the isolation of the ll-hydroxylated steroid from the broth mixture. The chloroform is then evaporated off in vacuo and the residue is recrystallized from acetone-hexane, to

24 yield IGuL-mGlhYlHIlOPIGgHElI'IC-Ila,l7a,2I-lriOl-3,20-Cll0n, after being chromatographed and eluted from a column as described in Example 3.

EXAMPLE 23 1 o'fl-melh y lallopregrmne-I I a, 1 711,21 -tri0l-3 ,ZO-dione Agar slants containing medium No. 1 described above and 1.5% by weight agar are sterilized for 15 minutes at 121 C. at a pressure of 15 pounds p.s.i. The agar slants are then cooled to about 28 C. slanted and inoculated with a vegetative growth of a culture Glomerclia fusariaides (ATCC 9552) and incubated at a temperature of 28 C. until heavy sporulation occurs.

A two liter Erlenmeyer flask containing 500 ml. of a similarly sterilized and cooled broth of medium No. l is then inoculated with spores from one of the heavily sporulated agar slants and incubated from 24 hours to 36 hours at 28 C. on a New Brunswick rotary shaker set at 280 rpm. At the end of this period 500 mg. of 16,8-methylallopregnane-1711.21-diol-3,20 dione. prepared as described in Example 15(a-e), in 5 ml. of ethanol is added. The flask is replaced on the rotary shaker and incubation continued for a period of from 24 hours to 30 hours during which latter period 10 ml. samples re moved at intervals from the Erlenmeyer flask and extracted with chloroform are then chromatographcd on paper (according to the method of Bush as modified by Shull) employing a toluene/ethyl acetate solvent system, the paper being impregnated with acetone and water. Incubation is continued until chromatography indicates complete transformation of lofi-methyialiopregnane 17a,21-diol-3.20-dione to 16B-methylallopregnane-lla; l7a,2ltriol-3.20-dione. The broth mixture in the Er enmeyer flask is then extracted with chloroform to effect the isolation of the llhydroxylated steroid from the broth mixture. The chloroform is then evaporated off in vacuo and the residue is recrystallized from acetone-hexane to yield lfidmethylallopregnaned 112.17%,21 -triol 3 .ZO-dione. after being chromatographed and eluted from a column as described in Example 3.

EXAMPLE 24 l6a-tert.-butylallopregnane-I1a,] 711,2] triol-3,20-dione Agar slants containing medium No. 1 described above and 1.5% agar are sterilized for 15 minutes at 121 C. at a pressure of 15 pounds psi. The agar slants are then cooled to about 28 C. slanted and inoculated with a vegetative growth of a culture Glomerella glycines (Baarn) and incubated at a temperature of 28 C. until heavy sporulation occurs.

A two liter Erlenmeyer flask containing 500 ml. of a similarly sterilized and cooled broth of medium No. 1 is then inoculated with spores from one of the heavily sporulated agar slants and incubated from 24 hours to 36 hours at 28 C. on a New Brunswick rotary shaker set at 280 r.p.m. At the end of this period 500 mg. of 16vt-tert.-butylallopregnane ;.21 diol-3,20-dione. prepared as described in Example 16(a-e), in 5 ml. of ethanol is added. The flask is replaced on the rotary shaker and incubation continued for a period of from 24 hours to 30 hours during which latter period 10 ml. samples removed at intervals from the Erlenmeyer flask and extracted with chloroform are then chromatographed on paper (according to the method of Bush as modified by Shull) employing a toluene/ethyl acetate solvent system, the paper being impregnated with acetone and water. Incubation is continued until chromatography indicates complete transformation of l6a-terL-butylaliopregnanel7a,2l-dl0l-3,20 dione to 16:1 tert.-butylallopregnanel1a,17a,2l-triol-3,20-dione. The broth mixture in the Erlenmeyer flask is then extracted with chloroform to effect the isolation of the ll-hydroxylated steroid from the broth mixture. The chloroform is then evaporated otf in vacuo and the residue is recrystallized from ace tone-hexane to yield l6a-tert.-=butylallopregnane-11u,17ct,-

2l-triol-3,20-dione, after being chromatographed and eluted from a column as described in Example 3.

EXAMPLE 25 I 6 fl-ethylaIIopregnane-I 1 11,1 701,21 -triI-3,20-dione Agar slants containing medium No. 1 described above and 1.5% agar are sterilized for 15 minutes at 121 C. at a pressure of 15 pounds p.s.i. The agar slants are then cooled to about 28 C. slanted and inoculated with a vegetative growth of a culture Glomerella lagenarium (Baarn) and incubated at a temperature of 28 C. until heavy sporulation occurs.

A two liter Erlenmeyer flask containing 500 ml. of a similarly sterilized and cooled broth of medium No. 1 is then inoculated with spores from one of the heavily sporulated agar slants and incubated from 24 hours to 36 hours at 28 C. on a New Brunswick rotary shaker set at 280 r.p.m. At the end of this period 500 mg. of 16fi-ethylallopregnane-170:,21 diol-3,20-dione, prepared as described in Example 17 (0-2), in 5 ml. of ethanol is added. The flask is replaced on the rotary shaker and incubation continued for a period of from 24 hours to 30 hours during which latter period ml. samples removed at intervals from the Erlenmeyer flask and extracted with chloroform are then chromatographed on paper (according to the method of Bush as modified by Shull) employing a toluene/ethyl acetate solvent system, the paper being impregnated with acetone and water. Incubation is continued until chromatography indicates complete transformation of l65-ethylallopregnane-17a,- 2l-diol-3,20-dione to lfifl-ethylallopregnane-l1a,17a.21- triol-3,20-dione. The broth mixture in the Erlenmeyer flask is then extracted with chloroform to effect the isolation of the ll-hydroxylated steroid from the broth mixture. The chloroform is then evaporated oil in vacuo and the residue is recrystallized from acetone-hexane to yield 16/S-ethylallopregnane 1la,l7a,2l-triol-3,20-dione, after being chromatographed and eluted from a column as described in Example 3.

EXAMPLE 26 16a-methyl-I ,4-pregnadiene-11aJ 7a,21-triol-3,20-di0ne Agar slants containing medium No. 1 described above and 1.5 by weight agar are sterilized for 15 minutes at 121 C. at a pressure of 15 pounds p.s.i. The agar slants are then cooled to about 28 C. slanted and inocu lated with a vegetative growth of a culture Glomerella cingnlam (ATCC 10533) and incubated at a temperature of 28 C. until heavy sporulation occurs.

A two liter Erlenmeyer flask containing 500 ml. of a similarly sterilized and cooled broth of medium No. l is then inoculated with spores from one of the heavily sporulated agar slants and incubated from 24 hours to 36 hours at 28 C. on a New Brunswick rotary shaker set at 280 r.p.m. At the end of this period 500 mg. of l6a-methyl-l,4-pregnadiene 17a,2l-diol-3,20-dione, prepared as described in Example 18(a, b), in 5 ml. of ethanol is added. The flask is replaced on the rotary shaker and incubation continued for a period of from 24 hours to 30 hours during which latter period 10 ml. samples removed at intervals from the Erlenmeyer flask and extracted with chloroform are then chromatographed on paper (according to the method of Bush as modified by Shull) employing a toluene/ethyl acetate solvent system. the paper being impregnated with acetone and water. Incubation is continued until chromatography indicates complete transformation of l6u-methyl-l,4-pregnadiene- 17a,21-di0l3,20 dione to 16st methyl-1,4-pregnadienella,l7a,2l-triol-3,20-dione. The broth mixture in the Erlenmeyer flask is then extracted with chloroform to effect the isolation of the ll-hydroxylated steroid from the broth mixture. The chloroform is then evaporated off in vacuo and the residue is recrystallized from acetone to yield 16a-methyl-1,4 pregnadiene 11e,17a,21-triol- 26 3,20-dione, after being chromatographed and eluted from a column as described in Example 3.

Agar slants containing medium No. 1 described above and 1.5% by weight agar are sterilized for 15 minutes at 121 C. at a pressure of 15 pounds p.s.i. The agar slants are then cooled to about 28 C. slanted and inoculated with a vegetative growth of a culture GlomereIIa major (Baarn) and incubated at a temperature of 28 C. until heavy sporulation occurs.

A two liter Erlenmeyer flask containing 500 ml. of a similarly sterilized and cooled broth of medium No. l is then inoculated with spores from one of the heavily sporulated agar slants and incubated from 24 hours to 36 hours at 28 C. on a New Brunswick rotary shaker set at 280 r.p.m. At the end of this period 500 mg. of ldfi-methyl-1,4-pregnadiene-17a,2l-diol-3,20 dione, prepared as described in Example 19(a, b), in 5 ml. of ethanol is added. The flask is replaced on the rotary shaker and incubation continued for a period of from 24 hours to 30 hours during which latter period 10 ml. samples removed at intervals from the Erlenmeyer flask and extracted with chloroform are then chromatographed on paper (according to the method of Bush as modified by Shull) employing a toluene/ethyl acetate solvent system, the paper being impregnated with acetone and water. Incubation is continued until chromatography indicates complete transformation of 16fl-methyl-1,4-pregnadiene- 17a,21-diol-3,20-dione to 1fifi-methyl-1,4-pregnadienella,l7a,21-triol-3,20-dione. The broth mixture in the Erlenmeyer flask is then extracted with chloroform to effect the isloation of the ll-hydroxylated steroid from the broth mixture. The chloroform is then evaporated off in vacuo and the residue is recrystallized from acetone to yield 16,8 methyl 1,4-pregnadiene-l1m,I7a,2l-triol-3,20- dione, after being chromatographed and eluted from a column as described in Example 3.

EXAMPLE 28 1 Ga-terL-butyl-I ,4-pregnadiene-I 1 01,1 70:,21 -tri0l,3,20-

dione Agar slants containing medium No. 1 described above and 1.5% agar are sterilized for 15 minutes at 121 C. at a pressure of 15 pounds p.s.i. The agar slants are then cooled to about 28 C. slanted and inoculated with a vegetative growth of a culture Glomerella phat-idiomorpha (Baarn) and incubated at a temperature of 28 C. until heavy sporulation occurs.

A two liter Erlenmeyer flask containing 500 ml. of a similarly sterilized and cooled broth of medium No. 1 is then inoculated with spores from one of the heavily sporulated agar slants and incubated from 24 hours to 36 hours at 28" C. on a New Brunswick rotary shaker set at 280 r.p.m. At the end of this period 500 mg. of l6a-tert.-butyl-l,4-pregnadiene-l7e,2l diol 3,20 dione, prepared as described in Example 20(a, b), in 5 ml. of ethanol is added. The flask is replaced on the rotary shaker and incubation continued for a period of from 24 hours to 30 hours during which latter period 10 ml. samples removed at intervals from the Erlenmeyer flask and extracted with chloroform are then chromatographed on paper (according to the method of Bush as modified by Shull) employing a toluene/ethyl acetate solvent system, the paper being impregnated with acetone and water. Incubation is continued until chromatography indicates complete transformation of l6u-tert.-butyl-l,4- pregnadiene-17a,2l-diol-3,20-dione to l6a-tert.-butyl-l,4- pregnadiene-lla,17a,21-triol-3,20-dione. The broth mixture in the Erlenmeyer flask is then extracted with chloroform to eifect the isolation of the ll-hydroxylated steroid from the broth mixture. The chloroform is then evaporated oil in vacuo and the residue is recrystallized from acetone to yield 16m-tert.-butyl-1,4-pregnadiene-1la,17a,

assesses 21-triol-3,20-dione, after being chromatographed and eluted from a column as described in Example 3.

EXAMPLE 29 I 6fl-ethyl-I ,4 -pregnadiene-1 1 01,1 7a,21 -triol-3,20-dine Agar slants containing medium No. 1 described above and 1.5% agar are sterilized for 15 minutes at 121 C. at a pressure of 15 pounds p.s.i. The agar slants are then cooled to about 28 C. slanted and inoculated with 1 a vegetative growth of a culture Glomerella cingulata (ATCC 10531) and incubated at a temperature of 28 C. until heavy sporulation occurs.

A two liter Erlenmeyer flask containing 500 ml. of a similarly sterilized and cooled broth of medium No. 1 is then inoculated with spores from one of the heavily sporulated agar slants and 10 ml. of anti-foam, Larex (1 percent octadccanol in lard oil) added to the culture medium and incubated from 24 hours to 36 hours at 28 C. on a New Brunswick rotary shaker set at 280 r.p.m. At the end of this period 500 mg. of 16fi-ethyl-1A-pregnadiene-l7a,21-diol-3,20 dione, prepared as described in Example 21(a, b), in ml. of ethanol is added. The flask is replaced on the rotary shaker and incubation continued for a period of from 24 hours to 30 hours during which latter period 10 ml. samples removed at intervals from the Erlenmeyer flask and extracted with chloroform are then chromatographed on paper (according to the method of Bush as modified by Shull) employing a toluene/ ethyl acetate solvent system, the paper being impregnated with acetone and water. Incubation is continued until chromatography indicates complete transformation of 165-ethyl-1,4-pregnadiene-17a,21-diol-3,20- dione to 16p-ethyl'l,4-pregnadiene-11a,17a,21-triol-3,20- dione. The broth mixture in the Erlenmeyer flask is then extracted with chloroform to efiect the isolation of the ll-hydroxylated steroid from the broth mixture. The chloroform is then evaporated oif in vacuo and the residue is recrystallized from acetone-hexane to yield 166- ethyl-l,4-pregnadiene-l1a,17a-21-triol 3,20 dione, after being chromatographed and eluted from a column as described in Example 3.

EXAMPLE 30 16a-methyl-4-pregnene-I 1a,] 7a,21 -triol-3,20-dione (n) 1swMETHYL-neDICnLORO-PREGNANEee-On 20-ONE-3ACETATE A solution containing 5.6 g. (0.0155 mole) of 16amethylpregnenolone-3-acetate in 5 ml. of pyridine and 150 ml. of carbon tetrachloride is cooled to -23 C. To this stirred solution is added dropwise over a ten minute period 1.16 g. (0.016 mole) of chlorine contained in 20 ml. of carbon tetrachloride. The reaction solution is then allowed to warm to approximately C. while maintaining the stirring. Methylene chloride (70 ml.) is then added and the solution is washed successively with dilute hydrochloric acid, water, aqueous sodium bicarbonate, and then water. The organic phase is dried over magnesium sulfate, filtered, and the filtrate evaporated to near dryness, and treated with methanol, whereupon there is obtained 1Ga-methyl-S,6-dichloro-pregnane-3fl-ol- -one-3-acetate, M.P. 195-196.

(h) 1 0a-METHYL-D,B-DICIILORO-PREGNANE-3.l 7n'DIOL- 20-ONE-3-l tCETATE To 125 ml. of acetic anhydride is added 5.0 g. (0.0111 mole) of 16ormethyl-5,6-dichloro-pregnane-3B-ol-20-one' 3-acetate and 1.0 g. (0.0053 mole) of p-toluene sulfonic acid monchydrate. The solution is refluxed for 6 hours while maintaining a constant distillation rate so that there is collected during this time 100 ml. of distillate. The reaction solution is cooled and poured into 400 ml. of water and the mixture stirred in order to facilitate the hydrolysis of the acetic anhydride. The aqueous mixture is extracted two times with 100 ml. portions of benzene. The combined benzene solution is washed two times with 50 ml. portions of water and one time with 50 ml. of a 2% solution of sodium acetate in water. The organic phase is dried over magnesium sulfate and filtered. The filtrate is concentrated under vacuum to a volume of 70 ml. of benzene and then stirred for 19 hours with a mixture of 0.52 g. of sodium acetate in 12 ml. of commercial 40% peracetic acid. Excess peracetic acid is then destroyed by the dropwise addition of a solution of 15.6 g. of sodium sulfite in 53 ml. of water, while maintaining the 0 temperature between 1020 C. An additional 1.7 g. of

sodium sulfite is then added, and the mixture is stirred until a starch-iodide test is negative. The benzene layer is separated, washed three times with water, and evaporated. To the residue is added 200 ml. of methanol and 20 ml. of water containing 6.2 g. of potassium bicarbonate. The reaction solution is refluxed for 2 hours, and after the addition of 4 ml. of acetic acid, is concentrated under vacuum to a volume of 40 ml., which is distilled with 20 volumes of water. Separation of the resultant precipitate and crystallization from ethyl acetate affords 16ot-methyl-5,6'dichloro-3ft,17a-diol-20-one, M.P. 216-217 C.

(c) 1(Se-METHYL-5,B-DICHLORO-PREGNANE-ZO-ONE- 35.17a.21-TRIOL-21-ACETATE To a solution containing 1.0 g. (0.0024 mole) of 16amethyl-S,6-dichloro-pregnane-ZO-one-3B,17a-diol-20 one in 50 ml. of C.P. chloroform (containing a few drops of chloroform previously saturated with hydrogen bromide) maintained at 20 C. is added (over a 20 minute-period) 0.396 g. of bromine in 6 ml. of chloroform. The solution is stirred an additional 20 minutes and then washed 3 times with water, dried over magnesium sulfate, and filtered. The filtrate is concentrated under vacuum to 20 ml. and stirred at 45 C. with 20 ml. of methanol and 0.72 g. (0.0048 mole) of sodium iodide for 1 hour and thirty minutes. Water is added and the mixture extracted with chloroform. The combined chloroform extracts are washed with water, dried over magnesium sulfate, and filtered. The filtrate is evaporated to dryness and the residue dissolved in 40 ml. of acetone and 2 ml. of water containing 0.72 g. (0.0063 mole) of potassium acetate. The solution is refluxed for 18 hours, evaporated to near dryness and water added. The resultant precipitate is filtered, washed with water, and then crystallized from isopropanol to obtain 16amethyl-5,6-dichloro-pregnane- 20-one-3B,l7a,21-triol 21-acetate.

a) 16aMETHYL-5,6-DICHLORO-PREGNANE-l7e21- DIGLJLZO'DIONE To a stirred solution of 1.5 g. of 16a-methyl-5,6-dichloro-pregnane-20-one-3fl,17a,21-triol-2l-acetate in 40 ml. of acetic acid and 4 ml. of water maintained at 10 C. is first added over a 20-minute period a solution containing 0.34 g. of chromium trioxide in 5 ml. of acetic acid and 0.5 ml. of water, and then over a four-minute period 0.19 ml. of concentrated sulfuric acid. The reaction mixture is stirred for two hours, then diluted with water, and extracted with chloroform. The combined chloroform extracts (150 ml.) are washed successively with water (70 ml.) three times with a 3% sodium bicarbonate solution and finally with 60 ml. of water. The chloroform solution is dried over magnesium sulfate and evaporated to dryness. The residue upon crystallization from acetone-hexane affords 16a-methyl-5,6-dichloro-pregnane- 17a,2l-dio1-3,20dione.

(e1 16a-METHYL-4-PREGNENEJ7:1,21-DI0L-320-DI0NE ZLACETATE To a stirred solution of 1.5 g. of 16a-methyl-5,6-dichloro-pt'egnane-l7a,21-diol-3,20-dione 21-acetate in 100 ml. of acetic acid maintained at C. there is added 1 g. of zinc dust, followed after 45 minutes by an additional gram of zinc dust. After another 45 minutes at 75 the reaction solution is filtered to remove the insoluble zinc. The filtrate is air-evaporated to approximately 5 ml. and then diluted with 30 ml. of water. The resultant precipitate is filtered, washed with water, and after crystallization from acetone-hexane atfords l6e-methyl-4-pregnene-l7a,21-diol-3,20-dione 21-acetate.

(I J 1 6a-METHYL-4-PREGNENE-17a,21-DIOL-3,20-DIONE Method I.-One gram of 16a-methyl-4-pregnene-17a, 21-diol-3,20-dione 21-acetate is dissolved in 25 ml. of methanol and 5 ml. of water containing 0.19 g. of potassium bicarbonate. This solution is refluxed for 35 minutes and then after the addition of 0.12 ml. of acetic acid is concentrated under vacuum to a residue, to which 15 ml. of water is added. The resulting precipitate is filtered and dried. Crystallization from acetone-hexane gives 16a-methyl-4-pregnene-17a,21diol-3,20-dione.

Method II.--Alternatively, 16a-methyl-4-pregnene-17a, 2l-diol-3,20-dione is prepared from 16tx-methyl-5,6-diehloro-pregnane-20-one-3p,17a,2l-triol-2l-acetate as follows:

To a solution of 1.0 g. of l6u-methyl-5,6-dichloro-pregnane-20-one-3p,17a,21-triol 2l-acetate in 150 ml. acetone under an atmosphere of carbon dioxide there is added a solution of chromous chloride (prepared from 40 g. of amalgamated zinc dust, 8 m1. concentrated hydrochloric acid, 80 ml. water and 20 g. chromic chloride). After standing at room temperature for two hours, 20 ml. of water is added and the solution is evaporated to approximately 10 ml., diluted with 30 ml. water, and extracted 3 times with 30 ml. portions of methylene chloride. The combined methylene chloride extracts are washed with water and then dried over magnesium sulfate and filtered. The filtrate is evaporated to a crystalline residue, which upon treatment with acetone-hexane gives IGa-methyI-S- pregnene-20-one-3fl,17e-21-triol-21-acetate, which is then employed in the following procedure:

A medium having a composition of 10 grams of yeast extract (Difco), 4.5 g. of potassium dihydrogen phos phate and 4.7 g. of disodium hydrogen phosphate monohydrate is diluted to 1 liter with tap water, dispersed in aliquots of 100 ml. into 300 ml. Erlenmeyer flasks and sterilized for 20 minutes at pounds steam pressure. The pH after sterilization is 6.8.

The sterile medium in the flasks is inoculated with an agar slant of Flavobacterium dehydrogenans var. hydrolyticum classified in the Rutgers Collection as No. 130, Rutgers University, New Brunswick, New Jersey, or with 1% by volume of a 24-hour broth culture. The inoculated flask is placed in a shaking machine set at 248 strokes per minute, in an incubator kept at 30 C. The shake cultures are subjected to continuous illumination.

Twelve to twenty-four hours later, 200 mg. of 16amethyl-5-pregnene-20one-3/3,17a,21-triol ZI-acetate dissolved in 5 ml. of 95% ethanol is added to each flask. The pH is now 7.2-7.4.

After 60 hours of shaking, the fermentation is stopped. The final pH is 7.5-7.8. The pH is then adjusted to 3.5 with hydrochloric acid and the fermentation liquors autoclaved for 15 mrninutes at 15 pounds steam pressure. After cooling, the broth is filtered with the aid of 2% of Filter-Gel (i.e. diatomaceous earth). Both the filtrate and the filter cake are extracted thoroughly with chloroform and the combined extracts evaporated to dryness in vacuo. The combined residual solid from the ten flasks is crystallized from acetone-hexane to give 16: methyl-4-pregnene-17e,21-diol-3,20-dione.

Alternatively, 16a-methyl-5-pregnene-20-one-3fi,170:,21- triol-2l-acetate 1.9 g.) is dissolved in 200 ml. of acetone (distilled from permanganate) and cooled to 10-15 under an atmosphere of nitrogen. To this stirred solution is added rapidly, but dropwise, 1.4 ml. of standard chromium trioxide reagent (prepared from 13.36 g. of chromium trioxide in 11.5 ml. of concentrated sulfuric acid diluted with water to a volume of 50 ml.). After 5 minutes, water is added and the resulting precipitate was washed well with water. In this manner there is obtained 1fia-methyl-S-pregnene-l7a,2l-diol-3,20-dione. When 1.0 g. of this latter substance is treated with 0.3 g. of potassium bicarbonate in 40 ml. of methanol and 4 ml. of

Water at reflux for 35 minutes under a nitrogen atmosphere, there is obtained, after the usual work-up, the desired 16a-methy1-4-pregnene- :,2 1-diol-3,20-dione.

(g) lfia-METHYL-4-PREGNENEr11a,1 7u,21-TRIOL- 3,20DIONE Agar slants containing medium No. 1 described above and 1.5% agar are sterilized for 15 minutes at 121 C. at a pressure of 15 pounds p.s.i. The agar slants are then cooled to about 28 C. slanted and inoculated with a vegetative growth of a culture Glomerella cingulam (ATCC 10534) and incubated at a temperature of 28 C. until heavy sporulation occurs.

A two liter Erlenmeyer flask containing 500 ml. of a similarly sterilized and cooled broth and medium No. l is then inoculated with spores from one of the heavily sporulated agar slants and 10 ml. of anti-foam, Larex 1% octadecanol in lard oil) added to the culture medium, and incubated from 24 hours to 36 hours at 28 C. on a New Brunswick rotary shaker set at 280 rpm. At the end of this period 500 mg. of lfia-methyl- 4-prcgnene-17a,2l-diol-3,20-dione in 5 ml. of ethanol is added. The flask is replaced on the rotary shaker and incubation continued for a period of from 24 hours to 30 hours during which latter period 10 ml. samples removed at intervals from the Erlenmeyer flask and extracted with chloroform are then chromatographed on paper (according to the method of Bush as modified by Shull) employing a toluene/ethyl acetate solvent system, the paper being impregnated with acetone as a dispersant and water. Incubation is continued until chromatography indicates complete transformation of 16a-methyl-4- pregnene l7tx,2l diol 3,20 dione to l6a-methyl-4- pregnene-lla,l7a,21-triol-3,20-dione. The broth mixture in the Erlenmeyer flask is then extracted with chloroform to effect the isolation of the ll-hydroxylated steroid from the broth mixture. The chloroform is then evaporated off in vacuo and the residue is recrystallized from acetone to yield 160: methyl-4-pregnene-1la,17a,2l-triol-3,20- dione.

EXAMPLE 3 l l6fi-merhyl-4-pregnene-I1a,] 7a,21 -trioI-3,20-di0ne To a stirred solution of 395 g. (1.2 moles) of 16- methyl-S,l6-pregnadien 3p-ol-20-one in a mixture of 1200 ml. of chloroform and 3000 ml. of methanol was added 75.4 ml. of 50% sodium hydroxide, 300 ml. of water and 540 ml. of 30% hydrogen peroxide. After stirring for 72 hours at room temperature, 1500 ml. of water were added and the mixture was then neutralized with acetic acid. The organic solvents were removed by steam distillation and the crude product after filtration and drying weighed 386 g. (83.3%), M.P. l81186 5.8 (1% diox.). Ultra-violet adsorption spectrum showed the presence of less than 1% starting material. The crude product upon recrystallization from methanol showed no ultraviolet adsorption, M.P. 190 (Kofier hot bench).

A solution of 657 g. of the crude epoxy compound in 2000 ml. of dry pyridine and 657 ml. of acetic anhydride was kept at 60 C. for 4 hours. After approximately one hour, crystals of the acetate appeared. The acetylation mixture was poured into ice water and the product filtered and dried to give a yield of 712 g., M.P. 163l74. Recrystallization from acetone gave a product without ultraviolet adsorption; yield 557 g. (75.5%), M.P. 182, [ch l6.7 (1% dioxane).

31 (c) 3,8-ACETOXY-16-METHYLENE-Ei-PREGNEN-l(a-OL- 20-ONE To a stirred solution of 275 g. (0.71 mole) of 3B- acetoxy-l6B-methyl-l6a,17a-epoxy pregnene-ZO-one in 6000 ml. of glacial acetic acid at 27 C. was added 6.05 g. of HBr (0.075 mole) in 100 ml. of glacial acetic acid. Within one minute crystallization of the product ensued. After stirring an additional 5 minutes, the methylene compound was filtered, washed with 80% aqueous acetic acid and finally with water. The product was dried at 110 C. under vacuum to break the acetic acid solvate', yield 207 g. (76.4%), M.P. 200 Kofier hot bench, [011 ---109 (1% dioxane). The acetic acid mother liquor, not including the water wash, was treated with 8 g. of potassium acetate to neutralize the I-lBr present. The volume was reduced to approximately one liter and cooled to give an additional 34 g. of product, melting at 200 (Kofler hot bench), [11],; 95 (1% dioxane). This was an overall yield of 88.9% on the two fractions of 3;3-acetoxy-16-methylene 5 pregnene-l7a-ol-20-one. The sample for analysis was recrystallized from acetone, M.P. 199-201 C., 200 C. (Kofler hot bench) 1l0 (1% dioxane).

(a) BB-ACETOXY-l6fl-METHYL 5-PREGNEN-1la-0L- 204ONE A solution of 367 g. (0.95 mole) of the Bfi-acetoxyl6-methylene-5-pregnene-l7a-ol-20-one in 4.67 l. of tetrahydrofuran containing 59.7 ml. of triethylamine, was bydrogenated under 10 pounds pressure at 23 C. in the presence of 184 g. of palladium on carbon catalyst. After 100 minutes, the absorption of hydrogen stopped at 1 mole. The sterol solution, after removal of the catalyst, was concentrated under reduced pressure to a heavy slurry of crystals, and then 2.5 l. of hexane was added. The pure product, 3,9-acetoxy-l6B-methyl-5- pregnene-17a-ol-20-one was filtered and washed with hexane to give 315 g. (85.7%), MP. 168 C. (Kofler hot bench) (0:1 l7.99 (1% dioxane). The mother liquor upon concentration gave an additional 34 g. of pure product; MP. 169 (Kofier hot bench), [M335 --16.5 (1% dioxane). This was an overall yield of 94.5%.

The analytical sample was recrystallized from acetonehexane, M.P. l69l70 C. [111 20.6 (1% dioxane). Annlysis.--Found: C, 74.06, H, 9.2. Calcd: C, 74.30; H, 9.15.

(e) 16fi-METHYL-5-PREGNENE-3fi-17a-DIOL20-ONE To a refluxing solution of 50 g. of 3fl-acetoxy-l6fimethyl-S-pregnene-l7a-ol-20-one in 2500 ml. of methanol, was added 25 g. of sodium hydroxide dissolved in 250 ml. water. Crystallization took place almost immediately. The reaction was stirred at reflux for minutes and then acidified with 40 ml. of acetic acid. The slurry was poured into 4 l. of ice water, filtered, and washed neutral to give 43 g. (97%) of crude product melting with decomposition at 253260 (Kofler hot bench), [M1 16.8. Recrystallization from methanol gave 40.3 g. of 16,3-methyl-5-pregnene-3fi,l7a-diol-20-one melting at 260 (Kofler hot bench).

(f) 33,21pmcnroxraesiuntrnrn-sranosuma lTwOL-20-ONE A solution of 163 g. of bromine (1.02 moles) in 500 ml. of chloroform was added dropwise over a minute period to 194.3 g., 0.5 mole, of lfifl-methyl-S-pregnene- 3B-l7a-diol-20-one-3 acetate in 1750 ml. of chloroform with stirring at 2530 C. Stirring was continued for an additional 5 minutes when decolorization was complete. The solution was neutralized with 100 g. of solid sodium bicarbonate and filtered. The chloroform solution of the 5,6,2ltribrorno compound was concentrated to 1400 ml. under reduced pressure below 40 C. Methanol (1250 ml.) and 375 g. of sodium iodide were added and the mixture was stirred for one hour at 43 to 48 C. The reaction mixture was cooled to 10 C. by the addition of 3750 ml. of ice water and 125 g. of sodium bicarbonate was added. With good mechanical agitation 8.5% hydrazine hydrate solution was added dropwise until the iodine color was discharged. The hydrazine hydrate required (140 ml.) calculated to be 92% of the theroretical amount. The chloroform layer was separated from the aqueous phase and concentrated to almost dryness under reduced pressure below 40 C. The crude 21-iodo compound was then stirred 17 hours at refluxing temperatures with 1250 ml. of acetone, 250 ml. of water and 100 g. of potassium acetate. The acetone was then removed by steam distillation and the resulting crystalline product filtered and dried. The crude product was dissolved in 7 l. of ethyl ether, treated with decolorizing charcoal, and the product was finally crystallized from a mixture of ether-hexane. The product, 318,2l-diacetoxy-16B-methyl-5-pregnene-17m-ol-20-one was filtered and dried at C. under vacuum to give 127 g. (57%), M.P. 170 (Kofier hot bench), +15 1% dioxane).

(g) IBB-METHYL-l7ci,21-DIHYDROXY-4-PREGNENE- 3,20-DIONE A medium having a composition of 10 g. of yeast extract (Difco), 45 g. of potassium dihydrogen phosphate and 4.7 g. of disodium hydrogen phosphate monohydrate is diluted to 1 liter with tap water, dispersed in aliquots of ml. into 300 ml. Erlenmeyer flasks and sterilized for 20 minutes at 15 pounds steam pressure. The pH after sterilization is 6.8.

The sterile medium in the flasks is inoculated with agar slant of Flavobacterium dehydrogenans var. hydrolyticum (Rutgers Collection No. or with 1% by volume of a 24-hour broth culture. The inoculated flask is placed in a shaking machine set at 248 strokes per minute, in an incubator kept at 30 C. The shake cultures are subject to continuous illumination.

Twelve to twentyfour hours later, 200 mg. of 16:3- methyl-3fi,2l-diacetoxy 17a hydroxy-5-pregnen-20-one dissolved in 5 ml. of 95% ethanol is added to each flask. The pH is now 7.2 7.4.

After 60 hours of shaking, the fermentation is stopped. Reaction was followed by specific rotation until complete and the sterol extracted from the fermentation broth with ethyl acetate. The extracts were evaporated to dryness and sludged in 10 volumes of ethyl ether to remove impurities and unconverted starting material to give a substantially pure 1613-rnethyl-l7a,2l-dihydroxy-4-pregnene-3,20-dione, M.P. 214 C. Recrystallization from acetone-ether gives a pure product, M.P. 219 C.-220 C., [M +130 'C. (c.=l% dioxane).

n) 1.6fl-METHYL-110.,17a,2l-TRIHYDROXY- t-PREGNENE- 3,20-DIONE Agar slants containing medium No. 1 described above and 1.5% agar are sterilized for 15 minutes at 121 C. at a pressure of 15 pounds p.s.i. The agar slants are then cooled to about 28 C. slanted and inoculated with a vegetative growth of a culture Glomerella cingzilata (ATCC 10534) and incubated at a temperature of 28 C. until heavy sporulation occurs.

A two liter Erlenmeyer flask containing 500 ml. of a similarly sterilized and cooled broth of medium No. 1 is then inoculated with spores from one of the heavily sporulated agar slants and incubated from 24 hours to 36 hours at 28 C. on a New Brunswick rotary shaker set at 280 r.p.m. At the end of this period 500 mg. of l6fl-methyl-l7u,21-dihydroxy-4-pregnene-3,20-dione in 5 ml. of ethanol is added. The flask is replaced on the rotary shaker and incubation continued for a period of from 24 hours to 30 hours during which latter period 10 m1. samples removed at intervals from the Erlenmeyer flask and extracted with chloroform are then chromatographed on paper (according to the method of Bush as modified by Shull), employing a toluene/ethyl acetate solvent system, the paper being impregnated with acetone and water. Incubation is continued until chromatography indicates complete transformation of l6p-methyl-l7u,2ldihydroxy-4-pregnene-3,20-dione to IGB-methyl-lluglh, 21-trihydroxy-4-pregnene-3,20-dione. The broth mixture in the Erlenmeyer flask is then extracted with chloroform to effect the isolation of the ll-hydroxylated steroid from the broth mixture. The chlorofrom is then evaporated off in vacuo and the residue is recrystallized from acetone to yield pure lfifi-methyl-lla,l7a,2l-trihydroxy- 4-pregnene-3,20-dione, after being chromatographed and eluted from a column as described in Example 3.

This latter compound can be converted to l6/3-rnethyl- 1la,l7a,2l-trihydroxy-1,4-pregnadiene-3,ZO-dione by reaction with Corynebacterium simplex by the described procedure in United States Patent 2,837,464, issued June 3, 1958, to Arthur Nobile.

EXAMPLE 32 16fl-methyl-4-pregnene-1 I :,1 7a,21 -triol-3,20-dione (a) 21-ACETOXY-16B-METHYL5-PREGNENE-3fi.17a- DIOL-ZO-ONE A solution of 16.3 g. of bromine (0.102 mole) in 50 ml. of chloroform was added dropwise over a 30 minute period to 17.33 g. (0.05 mole) of 16,8-methyl-S- pregnene-3fl,l7a-dihydroxy-20-one in 1900 ml. of chloroform with stirring at 25-30" C. A small amount of hydrogen bromide gas was introduced at the start of the bromine addition to catalyze the bromination. Stirring was continued for an additional 10 minutes when decolorization was complete. The solution was neutralized with 50 g. of solid sodium bicarbonate and filtered. The chloroform solution of the 5,6,2l-tribromo compound was concentrated to 1400 ml. under reduced pressure below 40 C. Methanol (125 ml.) and 37.5 g. sodium iodide were added and the mixture was stirred for one hour at 43 to 48 C. The reaction mixture was cooled to 10 C. by the addition of 375 ml. of ice water and 125 g. sodium bicarbonate was added. With good mechanical agitation 8.5% hydrazine hydrate solution was added dropwise until the iodine color was discharged. The hydrazine hydrate required (14 ml.) calculated to be 95% of the theoretical amount for 0.05 mole of iodine. The chloroform layer was separated from the aqueous phase and concentrated to almost dryness under reduced pressure below 40 C. The crude 2l-iodo compound was then stirred 17 hours at refluxing temperatures with 125 ml. of acetone, 10 ml. of water and 10 g. of potassium acetate. The acetone was removed by steam distillation and the resulting crystalline product filtered. The wet filter cake was dissolved in methylene chloride, the water separated from the organic phase and the product was crystallized from a methylene chloridehexane mixture. The yield of compound, 2l-acetoxy lGfi-methyl-S-pregnene-SB,l7a-diol-20-one, was 16.0 g. (79.2% M.P. 170 (Kofler hot bench). After recrystallization from acetic acid and acetone-water, the pure compound melted at 175-476 C.

(b) 1 65-METHYL-17a-HYDROXY-21 -ACETOXY-4- PREGNENE-3,20-DIONE Seven grams of 1Gfl-methyl-S-pregnene-BB,l7a-diol-2lacetoxy-20-one was dissolved in 650 ml. of acetone, cooled to 10 C. and reacted with ml. of an aqueous solution of 1.33 g. chromium trioxide and 1.15 ml. of concentrated sulfuric acid. The chromium trioxide solution was added dropwise and the reaction blanketed with nitrogen. The reaction was stirred for an additional five minutes and then poured into 3.0 l. of ice water. After stirring for minutes, the precipitate was filtered, washed neutral with water and dried at 60 C. to give a yield of 6.0 grams, M.P. below 100 C., of S-keto-A compound. This was dissolved in tetrahydrofuran (50 ml.) and treated with 5 ml. of 6 N hydrochloric acid for two hours. The solvent is removed under reduced pressure and the residue crystallized from acetone-hexane to give the 16(3-methyl-I7e-hydroxy-2l-acetoxy-4-pregnene-3,20-dione.

(c) 16fl-METHYL-4-PREGNENE-11a,17a,21-TRIOL- 3,20 DIONE Agar slants containing medium No. 1 described above and 1.5% agar are sterilized for 15 minutes at 121 C. at a pressure of 15 pounds p.s.i. The agar slant is then cooled to about 28 C., slanted and inoculated with a vegetative growth of a culture Glomerella cingulata (QM 1407) and incubated at a temperature of 28 C. until heavy sporulation occurs.

A two liter Erlenmeyer flask containing 500 ml. of a similarly sterilized and cooled broth of medium No. l is then inoculated with spores from one of the heavily sporulated agar slants and incubated from 24 hours to 36 hours at 28 C. on a New Brunswick rotary shaker set at 280 r.p.m. At the end of this period 500 mg. of 16pmethyl 4 pregnene 1711,21 diol 3,20 dione 21- acetate in 5 ml. of ethanol is added. The flask is replaced on the rotary shaker and incubation continued for a period of from 24 hours to 30 hours during which latter period 10 ml. samples removed at intervals from the Erlenmeyer flask and extracted with chloroform are then chromatographed on paper (according to the method of Bush as modified by Shull) employing a toluene/ethyl acetate solvent system, the paper being impregnated with water together with acetone as a dispersant. Incubation is continued until chromatography indicates complete transformation of 16l9-methyl-4-pregnene-17a, 2l-diol-3,20-dione 2l-acetate to l6fi-methyl-4-pregnenella,l7a,2l-triol3,20-dione. The broth mixture in the Erlenmeyer flask is then extracted with chloroform to effect the isolation of the ll-hydroxylated steroid from the broth mixture. The chloroform is then evaported oil in vacuo and the residue is recrystallized from acetone-hexane to yield l6fi-methyl4-pregnene-11a,l7a,21- triol-3,20-dione, after being chromatographed and eluted from a column as described in Example 3.

What is claimed is:

l. A process which comprises subjecting an ll-desoxy steroid to the oxygenating activity of a species of the fungus of the genus Glomerella to cause the formation of the corresponding lla-hydroxylated derivative thereof.

2. A process which comprises subjecting an ll-desoxy steroid to the oxygenating activity of a species of the fungus of the genus Glomerella to produce the corresponding lla-hydroxylated derivative thereof.

3. A process which comprises subjecting an ll-desoxy steroid selected from the group consisting of a 3-ketopregnane, a 3-keto-alopregnane, and an unsaturated analogue thereof to the action of an oxygenating enzyme of a species of the fungus of the genus Glomerella to produce the corresponding lla-hydroxylated steroid derivative thereof.

4. The process of claim 1 wherein the fungus is Glomerella cingulata.

5. The process of claim 1 wherein the fungus is Glomerella fusarioid'es.

6. The process of claim 1 wherein the fungus is Glomeralla lagenarium.

7. The process of claim 1 wherein the fungus is Glomerella major.

8. The process of claim 1 wherein the fungus is Glomeralla phacidiomorpha.

9. The process of claim 1 wherein the fungus is G Iomerella rubicola.

10. The process of claim 1 wherein the ll-desoxy steroid is subjected to oxygenation under aerobic conditions.

11. A process which comprises subjecting a member selected from the group consisting of 4-pregnene-l7a, 2l-diol-3,20-dione and a C21 acylate thereof to the oxygenating fermentative action of a species of microorganism of the genus Glomerella to cause the formation of 4-pregnene-llm,l7a,2l-triol-3,20-dione.

12. A process which comprises subjecting 4-pregnenel7u,21-dio1-3,20-dione to the oxygenating fermentative action of the microorganism, Glomcrclhz cingula'ta to cause the formation of the corresponding Ila-hydroxylated derivative thereof.

13. A process which comprises subjecting the 21-acetate of 4-pregnene-l7ot,21-diol-3,20-dione to the oxygenating fermentative action of the microorganism Glnmcrelln cingulata to cause the formation of the corresponding lla-hydroxylated derivative thereof.

14. A process which comprises subjecting a member selected from the group consisting of 1,4-pregnadiene- 17a,21-diol-3,20-dione and a C-21 acylate thereof to the oxygenating fermentative action of a species of fungus of the genus Glomerella to cause the formation of 1,4- pregnadiene-l 1a,17a,21-triOl-3,Z(}di0n.

15. A process which comprises subjecting 1.4-pregnadiene-l7a,21-diol-3,20-dione to the oxygcnating fermentative action of the microorganism Glomerella cingulam to cause the formation of 1,4-pregnadiene-lla,l7a,2l-triol- 3,20-dione.

16. A process which comp-rises subjecting the C-2lacetate of 1,4-pregnadiene-17a 2l-diol-3,20-dione to the oxygenating fermentative action of the microorganism Glomerella cingulata to cause the formation of 1,4-pregnadiene-l1a,l7a,2l-triol-3,20-dione.

17. A process which comprises subjecting a member selected from the group consisting of 16a a1kyla]lopregnane-l7a,21-diol-3,20-dione and a C-2l-acetate thereof to the oxygenating fermentative action of a species of fungus of the genus Glomerella to cause the formation of 16a-alkylallopregnane-llm,17,2l-triol-3,20-dione.

18. A process which comprises subjecting mac-methylallopregnane-17u,2l-diol3,20-dione to the oxygenating fermentative action of the microorganism Glomerelin cingulata to cause the formation of l6a-methylallopregnane-1 lot,17a,21t1'i01-3,20-di011e.

19. A process which comprises subjecting the C-2lacetate of 16a-methylallopregnane-l7m,2l-diol-3,20-dione to the oxygenating fermentative action of the microorganism Glomerella cingulata to cause the formation of l6u-methylallopregnane-l lu,l7a,2l-triol-3 20-dione.

20. A process which comprises subjecting a member selected from the group consisting of lfifl-alkylallopregnane-l7a,2l-diol-3,20-dione and a C-2l-acylate thereof to the oxygenating fermentative action of a species of fungus of the genus Glomerella to cause the formation of the corresponding lla-hydroxylated derivative thereof.

21. A process which comprises subjecting l6 9-methyl' aI'lopregnane-17 2l-diol-3.20-dione to the oxygenating fermentative action of the microorganism Glomerclin cingulata to cause the formation of 16 S-methylal1opregnane-l 1a,170t,21-tl'i01-3 ,20-dione.

22. A process which comprises subjecting the C-Zlacetate of 1G S-methyIaIIQpregnane-l7a,2l-diol-3,20-dionc to the oxygenating fermentative action of the microorganism Glomerella cingulata to cause the formation of lfifi-methylallopregnane-l l :,17m2] -triol-3,20-dione.

23. A process which comprises subjecting l6a-tert. butylallopregnane-l7u,2l-diol-3,20-dione to the oxygenating fermentative action of the microorganism Glamerella cingulata to cause the formation of l6e-terL-butylallopregnane-l1a,17a,2l-triol-3,20-dione.

24. A process which comprises subjecting lfi-ethylallopregnane-l7a,2I-diol-3,ZO-dione to the oxygenating fermentative action of the microorganism Glomerella cingulata to cause the formation of l6fl-ethylalloprcgnane- 110:,17a,2l-t1'i0l-3,20-di0t1.

25. A process which comprises subjecting a member selected from the group consisting of a 16a-alkyl-L4- pregnadiene-l7a,2l-diol-3,20-dione and a C-2l-acylate thereof to the oxygenating fermentative action of a species of fungus of the genus Glomerella to cause the formation 36 of the corresponding lla-hydroxylated derivative thereof.

26. A process which comprises subjecting 16a-methyll,4-pregnadiene-l7a,2l-diol-3,20-dione to the oxygenating fermentative action of the microorganism Gi'omereila cingulata to cause the formation of I6a-rnethyI-L4-pregnadiene-l la,17a,21-triol-3,20-dionc.

27. A process which comprises subjecting the C 1 acetate of lout-methyl-1,4-pregnadiene-l7a,2l-diol-3,20- tlione to the oxygenating fermentative action of the microorganism Glomcrella cz'ngulata to cause the formation of ltia-rnethyl-l,4-pregnadiene-l lo,17ot,21-triol-3,20dione.

28. A process which comprises subjecting a member selected from the group consisting of a lp-alkyl-L-tpregnadiene-l7m,2l-diol-3,20-dione and a C-Zl-acylate thereof to the oxygenating fermcntative action of a species of fungus of the genus Glomerella to cause the formation of the corresponding lla-hydroxylated derivative thereof.

29. A process which comprises subjecting lop-methyl 1,4-pregnadiene-17a,21-diol-3,20-dione to the oxygenating fcrmentative action of a species of fungus of the genus Glomcrclla cingulata to cause the formation of lo 'l-methyl-l,4-pregnadienel la,17a,2l-triol-3,2()-dione.

30. A process which comprises subjecting the C2lacetate of lop-methyl-l,4-pregnadiene-l7a,2l-diol-3,20- dione to the oxygenating fcrmentative action of a species of fungus of the genus Glomerella cinguiaza to cause the formation of l6 3-methyl 1,4-pregnadiene-l lot,l7ot,2l-it'i01- 3,20-dione.

31. A process which comprises subjecting i6a-tCl't.- butylallopregnane-L4 pregnadiene 170:.21 diol 3,21)- dione to the oxygenating fermentative action of a species of fungus of the genus Glomerella cingulam to cause the formation of 1Gct-terL-butylalloprcgnane-l,4-prcgnadicncl la,17a,2l-triol-3,20-dione.

32. A process which comprises subjecting lop-ethyll,4-pregnadiene-17a,2l-diol-3,20dione to the ox /genating fermentative action of a species of fungus of the genus Glomerella cingulata to cause the formation of Mid-ethyl 1,4-pregnadiene-l 111,17u,2l-triol-3,20-dione.

33. A process which comprises subjecting a member selected from the group consisting of a l6-alkyl-4-pregnene-17a,21-diol-3,20-dione and a Zl-acylatc thereof to the oxygenatiug fermentative action of a species of fungus of the genus Glomerella to cause the formation of the corresponding llu-hydroxylated derivative thereof.

34. A process which comprises subjecting 16wmethy1' 4-pregnene 1701,21 diol 3,20 dione to the oxygenating fermentative action of a species of the genus Glomerella cingulata to cause the formation of l6a-methyl-4-pregnane-l 1a,17a,21-lriOl-3,20-di0n6.

35. A process which comprises subjecting the C-2lacetate of 16a-methyl-4-pregnene-l7a,2l-diol-3,20-dione to the oxygenating fermentative action of a species of fungus of the genus Glomerella cingulara to cause the formation of 16a-methy1-4-pregnane 11ct,17ct,2l triol- 3,20-dione.

36. A process which comprises subjecting 16fl-methyl- 4-pregnene 170:,21 diol 3,20 dione to the oxygenating fermentative action of a species of fungus of the genus Glomerelia cingulata to cause the formation of lfi-methyl-4-pregnane-1 1a,l7a,2l-triol-3,20-dione.

37. A process which comprises subjecting the C2lacetate of 165-rnethyl-4-pregnene-l7a,2l-dio1-3,20-dione to the oxygenating fermentative action of a species of fungus of the genus Glomerella cingulata to cause the formation of l6 3-methyl-4 pregnane 1la,17a,21 triol- 3,20-dione.

38. The process of claim 11 wherein the fungus is Glomerella Iagenarium.

39. The process of claim 14 wherein the fungus is G lomerella lagenarium.

40. The process of claim 25 wherein the fungus is Glomerella Iagenarium.

41. The process of claim 28 wherein the fungus is Glomerella Iagenarium.

42. The process of claim 33 wherein the fungus is Glomerella lagenarium.

43. The process which comprises the step of subjecting an ll-desoxy steroid selected from the group consisting of a pregnane, allopregnane, and an unsaturated analogue thereof to the enzymatic action of a species of fungus of the genus Glomerella to cause the formation of the 110:- hydroxylated derivative of said steroid and recovering said hydroxylated derivative therefrom.

44. A process which comprises the steps of subjecting an ll-desoxy steroid selected from the group consisting of a pregnane, allopregnane, and an unsaturated analogue thereof, to the enzymatic action of the fungus Glomerella cingulam to cause the formation of the lla-hydroxylated derivative of said steroid and recovering said hydroxylated derivatives therefrom.

45. A process which comprises the steps of inoculating a nutrient medium containing assimilable carbon, nitrogen and mineral salts and an ll-desoxy steroid selected from the group consisting of a pregnane, allopregnane, and an unsaturated analogue thereof with a fungus of the genus Glomerella and permitting the fermentation to proceed until a substantial amount of lla-hydroxylated derivative thereof has been formed.

46. The process of claim Glomerella cingulata.

47. The process of claim Glomerella fusarioides.

48. The process of claim 45 Glomerella lagenarium.

49. The process of claim Glomerella major.

50. The process of claim Glomerella phacidiomorpha.

S1. The process of claim Glomerella rubicola.

wherein the fungus is 45 wherein the fungus is wherein the fungus is 45 wherein the fungus is 45 wherein the fungus is 45 wherein the fungus is References Cited in the file of this patent UNITED STATES PATENTS Murray et a1 July 8, 1952 2,695,260 Murray et a] Nov. 23, 1954 

1. A PROCESS WHICH COMPRISES SUBJECTING AN 11-DESOXY STEROID TO THE OXYGENATING ACTIVITY OF A SPECIES OF THE FUNGUS OF THE GENUS GLOMERELLA TO CAUSE THE FORMATION OF THE CORRESPONDING 11A-HYDROXYLATED DERIVATIVE THEREOF. 